Direct Reprogramming of Human Suspension Cells into Mesodermal Cell Lineages via Combined Magnetic Targeting and Photothermal Stimulation by Magnetic Graphene Oxide Complexes

Suspension cells can provide a source of cells for cellular reprogramming, but they are difficult to transfect by nonviral vectors. An efficient and safe nonviral vector (GO‐Fe₃O₄‐PEI complexes) based on iron oxide nanoparticle (Fe₃O₄)‐decorated graphene oxide (GO) complexed with polyethylenimine (PEI) for the first time is developed for delivering three individual episomal plasmids (pCXLE‐hOCT3/4‐shp53, pCXLE‐hSK, and pCXLE‐hUL) encoding pluripotent‐related factors of Oct3/4, shRNA against p53, Sox2, Klf4, L‐Myc, and Lin28 into human peripheral blood mononuclear cells (PBMCs) simultaneously. The combined treatment of magnetic stirring and near‐infrared (NIR)‐laser irradiation, which can promote contact between the complexes and floating cells and increase the cell membrane permeability, respectively, is used to conduct multiple physical stimulations for suspension PBMCs transfection. The PCR analysis shows that the combinatorial effect of magnetic targeting and photothermal stimulation obviously promoted the transfection efficiency of suspension cells. The transfected cells show positive expression of the pluripotency markers, including Nanog, Oct4, and Sox2, and have potential to differentiate into mesoderm and ectoderm cells. The results demonstrate that the GO‐Fe₃O₄‐PEI complex provides a safe, convenient, and efficient tool for reprogramming PBMCs into partially induced pluripotent stem cells, which are able to rapidly transdifferentiate into mesodermal lineages without full reprogramming.     

Real‐Time Imaging of Dynamic Cell Reprogramming with Nanosensors

Cellular reprogramming, the process by which somatic cells regain pluripotency, is relevant in many disease modeling, therapeutic, and drug discovery applications. Molecular evaluation of reprogramming (e.g., polymerase chain reaction, immunostaining) is typically disruptive, and only provides snapshots of phenotypic traits. Gene reporter constructs facilitate live‐cell evaluation but is labor intensive and may risk insertional mutagenesis during viral transfection. Herein, the utilization of a non‐integrative nanosensor is demonstrated to visualize key reprogramming events in situ within live cells. Principally based on sustained intracellular release of encapsulated molecular probes, nanosensors successfully monitored mesenchymal‐epithelial transition, pluripotency acquisition, and transdifferentiation events. Tracking the dynamic expression of four pivotal biomarkers (i.e., THY1, E‐CADHERIN, OCT4, and GATA4 mRNA), nanosensor signal showed great agreement with polymerase chain reaction and gene reporter imaging (R² > 0.9). Overall, such facile, versatile nanosensor enables real‐time monitoring of low‐frequency reprogramming events, thereby useful for high‐throughput assessment, optimization, and biomarker‐specific cell enrichment.     

Generation of Integration‐Free Induced Neurons Using Graphene Oxide‐Polyethylenimine

Direct conversion of somatic cells into induced neurons (iNs) without inducing pluripotency has great therapeutic potential for treating central nervous system diseases. Reprogramming of somatic cells to iNs requires the introduction of several factors that drive cell‐fate conversion, and viruses are commonly used to deliver these factors into somatic cells. However, novel gene‐delivery systems that do not integrate transgenes into the genome are required to generate iNs for safe human clinical applications. In this study, it is investigated whether graphene oxide‐polyethylenimine (GO‐PEI) complexes are an efficient and safe system for messenger RNA delivery for direct reprogramming of iNs. The GO‐PEI complexes show low cytotoxicity, high delivery efficiency, and directly converted fibroblasts into iNs without integrating factors into the genome. Moreover, in vivo transduction of reprogramming factors into the brain with GO‐PEI complexes facilitates the production of iNs that alleviated Parkinson's disease symptoms in a mouse model. Thus, the GO‐PEI delivery system may be used to safely obtain iNs and could be used to develop direct cell reprogramming‐based therapies for neurodegenerative diseases.     

Self‐Assembling Proteins as High‐Performance Substrates for Embryonic Stem Cell Self‐Renewal

The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self‐renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large‐scale cell production under xeno‐free culture conditions using current matrices. Here, a bioactive, recombinant, protein‐based polymer, termed ZT^Fn, is presented that closely mimics human plasma fibronectin and serves as an economical, xeno‐free, biodegradable, and functionally adaptable cell substrate. The ZT^Fn substrate supports with high performance the propagation and long‐term self‐renewal of human embryonic stem cells while preserving their pluripotency. The ZT^Fn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.     

3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities

Human mesenchymal stem cells,such as human adipose-derived stem cells(hASCs),are typically cultured on a two-dimensional(2 D)monolayer material surface,on which 2 D culturing methods are easily performed and time-saving.However,hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2 D monolayer culturing method compared to hASCs cultured with a three-dimensional(3 D)culturing method,such as suspension culture.In this study,we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2 D and 3 D culturing processes.The hASCs cultivated with a 3 D culturing process after 2 D culture showed at least 2-fold enhanced pluripotency(Sox2,Nanog,and OCT4)compared with that of hASCs cultured with the 2 D culture process alone.Furthermore,hASCs obtained from the 3 D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2 D culture process when hASCs were induced to differentiate.However,their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3 D culture to 2 D culture and vice versa,which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2 D and 3 D culture.The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2 D and 3 D culturing processes.Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs,which may reduce the effects of the aging process in hASCs.We discuss whether the environment of stem cell culture(i.e.,2 D or 3 D cultivation)can affect stem cell fate in terms of pluripotency and differentiation reversibility.     

Fusion gene vectors allowing for simultaneous drug selection, cell labeling, and reporter assay in vitro and in vivo

Vector systems allowing simultaneously for rapid drug selection, cell labeling, and reporter assay are highly desirable in biomedical research including stem cell biology. Here, we present such a vector system including pCVpf or pCVpr, plasmids that express pf or pr, a fusion protein between puromycin acetyltransferase and green or red fluorescent protein from CV, the human cytomegalovirus enhancer/promoter. Transfection with pCVpf or pCVpr produced a ～10% efficiency of gene transfer. A 2-day pulse puromycin selection resulted in ～13-fold enrichment for transgenic cells, and continuous puromycin selection produced stable transgenic stem cell clones with retained pluripotency. Furthermore, we developed a PAC assay protocol for quantification of transgene expression. To test the usefulness for cell labeling and PAC assay in vivo, we constructed pVASpf containing pf linked to the regulatory sequence of medaka germ gene vasa and generated transgenic fish with visible GFP expression in germ cells. PAC assay revealed the highest expression in the testis. Interestingly, PAC activity was also detectable in somatic organs including the eye, which was validated by fluorescence in situ hybridization. Therefore, the pf and pr vectors provide a useful system for simultaneous drug selection, live labeling, and reporter assay in vitro and in vivo.     

Effective Gene Delivery into Human Stem Cells with a Cell‐Targeting Peptide‐Modified Bioreducible Polymer

Stem cells are poorly permissive to non‐viral gene transfection reagents. In this study, we explored the possibility of improving gene delivery into human embryonic (hESC) and mesenchymal (hMSC) stem cells by synergizing the activity of a cell‐binding ligand with a polymer that releases nucleic acids in a cytoplasm‐responsive manner. A 29 amino acid long peptide, RVG, targeting the nicotinic acetylcholine receptor (nAchR) was identified to bind both hMSC and H9‐derived hESC. Conjugating RVG to a redox‐sensitive biodegradable dendrimer‐type arginine‐grafted polymer (PAM‐ABP) enabled nanoparticle formation with plasmid DNA without altering the environment‐sensitive DNA release property and favorable toxicity profile of the parent polymer. Importantly, RVG‐PAM‐ABP quantitatively enhanced transfection into both hMSC and hESC compared to commercial transfection reagents like Lipofectamine 2000 and Fugene. ～60% and 50% of hMSC and hESC were respectively transfected, and at increased levels on a per cell basis, without affecting pluripotency marker expression. RVG‐PAM‐ABP is thus a novel bioreducible, biocompatible, non‐toxic, synthetic gene delivery system for nAchR‐expressing stem cells. Our data also demonstrates that a cell‐binding ligand like RVG can cooperate with a gene delivery system like PAM‐ABP to enable transfection of poorly‐permissive cells.     

Size‐Dependent Toxicity of Gold Nanoparticles on Human Embryonic Stem Cells and Their Neural Derivatives

This study explores the use of human embryonic stem cells (hESCs) for assessing nanotoxicology, specifically, the effect of gold nanoparticles (AuNPs) of different core sizes (1.5, 4, and 14 nm) on the viability, pluripotency, neuronal differentiation, and DNA methylation of hESCs. The hESCs exposed to 1.5 nm thiolate‐capped AuNPs exhibit loss of cohesiveness and detachment suggesting ongoing cell death at concentrations as low as 0.1 μg mL^?1. The cells exposed to 1.5 nm AuNPs at this concentration do not form embryoid bodies but rather disintegrate into single cells within 48 h. Cell death caused by 1.5 nm AuNPs also occur in hESC‐derived neural progenitor cells. None of the other nanoparticles exhibit toxic effects on the hESCs at concentrations as high as 10 μg mL^?1 during a 19 d neural differentiation period. Thiolate‐capped 4 nm AuNPs at 10 μg mL^?1 cause a dramatic decrease in global DNA methylation (5 mC) and a corresponding increase in global DNA hydroxymethylation (5 hmC) of the hESC's DNA in only 24 h. This work identifies a type of AuNPs highly toxic to hESCs and demonstrates the potential of hESCs in predicting nanotoxicity and characterizing their ability to alter the DNA methylation and hydroxymethylation patterns in the cells.     

Fluorescent nanoswitch for monitoring specific pluripotency-related microRNAs of induced pluripotent stem cells: Development of polyethyleneimine-oligonucleotide hybridization probes

The isolation of high-grade (i.e.high-pluripotency) human induced pluripotent stem cells (hiPSCs) is a decisive factor for enhancing the purity of hiPSC populations or differentiation efficiency.A non-invasive imaging system that can monitor microRNA (miRNA) expression provides a useful tool to identify and analyze specific cell populations.However,previous studies on the monitoring/isolation of hiPSCs by miRNA expression have limited hiPSCs' differentiation system owing to long-term incubation with miRNA imaging probe-nanocarriers.Therefore,we focused on monitoring high-grade hiPSCs without influencing the pluripotency of hiPSCs.We reduced nanoparticle transfection time,because hiPSCs are prone to spontaneous differentiation under external factors during incubation.The fluorescent nanoswitch ("ON" with target miRNA),which can be applied for either imaging or sorting specific cells by fluorescence signals,contains an miRNA imaging probe (miP) and a PEI-PEG nanoparticle (miP-P).Consequently,this nanoswitch can sense various endogenous target miRNAs within 30 min in vitro,and demonstrates strong potential for not only imaging but also sorting pluripotent hiPSCs without affecting pluripotency.Moreover,miP-P-treated hiPSCs differentiate well into endothelial cells,indicating that miP-P does not alter the pluripotency of hiPSCs.We envisage that this miRNA imaging system could be valuable for identifying and sorting high-grade hiPSCs for improved practical applications.     

Cellular Stemness Maintenance of Human Adipose‐Derived Stem Cells on ZnO Nanorod Arrays

Ever‐growing tissue regeneration and other stem cell therapies cause pressing need for large population of self‐renewable stem cells. However, stem cells gradually lose their stemness after long‐term in vitro cultivation. In this study, a ZnO nanorod (ZnO NR) array is used to maintain the stemness of human adipose‐derived stem cells (hADSCs). The results prove that after culturing hADSCs on ZnO NRs for 3 weeks, the stemness genes and protein expression level are higher than that on culture plates and ZnO film. ZnO NRs can maintain stemness of hADSCs without inhibiting the cell proliferation and oriented differentiation capabilities. KLF4 (Kruppel‐like factor 4) is a Zn²⁺‐binding gene that plays a vital role in cell proliferation and differentiation. Sustained Zn²⁺ release and the increased expression of KLF4 can be detected, suggesting that ZnO NRs have efficiently released Zn²⁺ for stemness maintenance. Taken together, the nanotopography of ZnO NRs and the Zn²⁺ release synergistically facilitate stemness maintenance. This study has provided a powerful tool for directing cell fate, maintaining stemness, and realizing the expansion of stem cells in vitro, which will open a new route for the manufacture of large populations of stem cells and fulfilling the growing demand for the cell therapy market.     

Quantitative Proteomics Study Reveals Changes in the Molecular Landscape of Human Embryonic Stem Cells with Impaired Stem Cell Differentiation upon Exposure to Titanium Dioxide Nanoparticles

The increasing number of nanoparticles (NPs) being used in various industries has led to growing concerns of potential hazards that NP exposure can incur on human health. However, its global effects on humans and the underlying mechanisms are not systemically studied. Human embryonic stem cells (hESCs), with the ability to differentiate to any cell types, provide a unique system to assess cellular, developmental, and functional toxicity in vitro within a single system highly relevant to human physiology. Here, the quantitative proteomics approach is adopted to evaluate the molecular consequences of titanium dioxide NPs (TiO₂ NPs) exposure in hESCs. The study identifies ≈328 unique proteins significantly affected by TiO₂ NPs exposure. Proteomics analysis highlights that TiO₂ NPs can induce DNA damage, elevated oxidative stress, apoptotic responses, and cellular differentiation. Furthermore, in vivo analysis demonstrates remarkable reduction in the ability of hESCs in teratoma formation after TiO₂ NPs exposure, suggesting impaired pluripotency. Subsequently, it is found that TiO₂ NPs can disrupt hESC mesoderm differentiation into cardiomyocytes. The study unveils comprehensive changes in the molecular landscape of hESCs by TiO₂ NPs and identifies the impact which TiO₂ NPs can have on the pluripotency and differentiation properties of human stem cells.     

诱导性多潜能干细胞(iPS)研究：现状与展望

通过特定的基因组合与转染可以将已分化的体细胞诱导重编程为多潜能干细胞(iPS),是近年来干细胞研究领域最令人瞩目的一项新的干细胞制造技术.与胚胎干细胞(ES)不同,iPS细胞的制造不需要毁损胚胎,因而不会涉及更多的伦理学问题.iPS的出现不仅为体细胞重编程去分化机制的研究注入了新的活力,而且为疾病发生发展相关机制研究与特异的细胞治疗,特别是再生医学带来新的曙光.目前,iPS的研究尚处于初级阶段,文章就iPS的研究现状与应用前景进行综述和展望.     

Mesenchymal Stem Cell Capping on ECM‐Anchored Caspase Inhibitor–Loaded PLGA Microspheres for Intraperitoneal Injection in DSS‐Induced Murine Colitis

Mesenchymal stem cells (MSCs) are considered as a promising alternative for the treatment of various inflammatory disorders. However, poor viability and engraftment of MSCs after transplantation are major hurdles in mesenchymal stem cell therapy. Extracellular matrix (ECM)‐coated scaffolds provide better cell attachment and mechanical support for MSCs after transplantation. A single‐step method for ECM functionalization on poly(lactic‐co‐glycolic acid) (PLGA) microspheres using a novel compound, dopamine‐conjugated poly(ethylene‐alt‐maleic acid), as a stabilizer during the preparation of microspheres is reported. The dopamine molecules on the surface of microspheres provide active sites for the conjugation of ECM in an aqueous solution. The results reveal that the viability of MSCs improves when they are coated over the ECM‐functionalized PLGA microspheres (eMs). In addition, the incorporation of a broad‐spectrum caspase inhibitor (IDN6556) into the eMs synergistically increases the viability of MSCs under in vitro conditions. Intraperitoneal injection of the MSC–microsphere hybrid alleviates experimental colitis in a murine model via inhibiting Th1 and Th17 differentiation of CD4⁺ T cells in colon‐draining mesenteric lymph nodes. Therefore, drug‐loaded ECM‐coated surfaces may be considered as attractive tools for improving viability, proliferation, and functionality of MSCs following transplantation.     

Artificially Reprogrammed Macrophages as Tumor‐Tropic Immunosuppression‐Resistant Biologics to Realize Therapeutics Production and Immune Activation

To engineer patient‐derived cells into therapy‐purposed biologics is a promising solution to realize personalized treatments. Without using gene‐editing technology, a live cell‐typed therapeutic is engineered for tumor treatment by artificially reprogramming macrophages with hyaluronic acid‐decorated superparamagnetic iron oxide nanoparticles (HIONs). This nanoparticle‐assisted cell‐reprogramming strategy demonstrates profound advantages, due to the combined contributions from the biological regulation of HIONs and the intrinsic nature of macrophages. Firstly, the reprogrammed macrophages present a substantial improvement in their innate capabilities, such as more effective tumor targeting and more efficient generation of bioactive components (e.g., reactive oxygen species, bioactive cytokines) to suppress tumor growth. Furthermore, this cell therapeutic exhibits cytostatic/proapoptotic effects specific to cancer cells. Secondly, HIONs enable macrophages more resistant to the intratumoral immunosuppressive environment. Thirdly, the macrophages are endowed with a strong ability to prime in situ protumoral M2 macrophages into antitumor M1 phenotype in a paracrine‐like manner. Consequently, a synergistic tumor‐inhibition effect is achieved. This study shows that engineering nanomaterial‐reprogrammed live cells as therapeutic biologics may be a more preferable option to the commonly used approaches where nanomaterials are administrated to induce bioresponse of certain cells in vivo.     

Magnetically Controlled Growth‐Factor‐Immobilized Multilayer Cell Sheets for Complex Tissue Regeneration

The scaffold‐free cell‐sheet technique plays a significant role in stem‐cell‐based regeneration. Furthermore, growth factors are known to direct stem cell differentiation and enhance tissue regeneration. However, the absence of an effective means to incorporate growth factors into the cell sheets hinders further optimization of the regeneration efficiency. Here, a novel design of magnetically controlled “growth‐factor‐immobilized cell sheets” is reported. A new Fe₃O₄ magnetic nanoparticle (MNP) coated with nanoscale graphene oxide (nGO@Fe₃O₄) is developed to label stem cells and deliver growth factors. First, the nGO@Fe₃O₄ MNPs can be easily swallowed by dental‐pulp stem cells (DPSCs) and have no influence on cell viability. Thus, the MNP‐labeled cells can be organized via magnetic force to form multilayered cell sheets in different patterns. Second, compared to traditional Fe₃O₄ nanoparticles, the graphene oxide coating provides plenty of carboxyl groups to bind and deliver growth factors. Therefore, with these nGO@Fe₃O₄ MNPs, bone‐morphogenetic‐protein‐2 (BMP2) is successfully incorporated into the DPSCs sheets to induce more bone formation. Furthermore, an integrated osteochondral complex is also constructed using a combination of DPSCs/TGFβ3 and DPSCs/BMP2. All these results demonstrate that the new cell‐sheet tissue‐engineering approach exhibits promising potential for future use in regenerative medicine.     

Mussel Adhesion‐Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose‐Derived Stem Cells

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution‐mediated transfection are limited due to low transfection efficiency and insufficient duration of cell‐siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio‐inspired polymer‐mediated reverse transfection system is developed consisting of implantable poly(lactic‐co‐glycolic acid) (PLGA) scaffolds functionalized with siRNA‐lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA‐coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA‐sLNP‐PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose‐derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA‐sLNP‐PLGA scaffolds enhanced bone formation in a mouse model of critical‐sized bone defect. Therefore, the bio‐inspired reverse transfection system can provide an all‐in‐one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.     

New Insights into Biocompatible Iron Oxide Nanoparticles: A Potential Booster of Gene Delivery to Stem Cells

Gene delivery to stem cells is a critical issue of stem cells‐based therapies, still facing ongoing challenges regarding efficiency and safety. Recent advances in the controlled synthesis of biocompatible magnetic iron oxide nanoparticles (IONPs) have provided a powerful nanotool for assisting gene delivery to stem cells. However, this field is still at an early stage, with well‐designed and scalable IONPs synthesis highly desired. Furthermore, the potential risks or bioeffects of IONPs on stem cells are not completely figured out. Therefore, in this review, the updated researches focused on the gene delivery to stem cells using various designed IONPs are highlighted. Additionally, the impacts of the physicochemical properties of IONPs, as well as the magnetofection systems on the gene delivery performance and biocompatibility are summarized. Finally, challenges attributed to the potential impacts of IONPs on the biologic behaviors of stem cells and the large‐scale productions of uniform IONPs are emphasized. The principles and challenges summarized in this review provide a general guidance for the rational design of IONPs‐assisted gene delivery to stem cells.     

The Regulatory Functionality of Exosomes Derived from hUMSCs in 3D Culture for Alzheimer's Disease Therapy

Reducing amyloid‐β (Aβ) accumulation could be a potential therapeutic approach for Alzheimer's disease (AD). Particular functional biomolecules in exosomes vested by the microenvironment in which the original cells resided can be transferred to recipient cells to improve pathological conditions. However, there are few reports addressing whether exosomes derived from cells cultured on scaffolds with varying dimension can reduce Aβ deposition or ameliorate cognitive decline for AD therapy. Herein, both 3D graphene scaffold and 2D graphene film are used as the matrix for human umbilical cord mesenchymal stem cell culture, from which the supernatants are obtained to isolate exosomes. The levels of 195 kinds of miRNAs and proteins, including neprilysin, insulin‐degrading enzyme and heat shock protein 70, in 3D‐cultured exosomes (3D‐Exo) are dramatically different from those obtained from 2D culture. Hence, 3D‐Exo could up‐regulate the expression of α‐secretase and down‐regulate the β‐secretase to reduce Aβ production in both AD pathology cells and transgenic mice, through their special cargo. With rescuing Aβ pathology, 3D‐Exo exerts enhanced therapeutic effects on ameliorating the memory and cognitive deficits in AD mice. These findings provide a novel clinical application for scaffold materials and functional exosomes derived from stem cells.