Genetic Modification Of Stem Cells Research Articles

Innovative approaches in stem cell therapy: revolutionizing cancer treatment and advancing neurobiology- A comprehensive review.

Stem cell therapy represents a transformative frontier in medical science, offering promising avenues for revolutionizing cancer treatment and advancing our understanding of neurobiology. This review explores innovative approaches in stem cell therapy that have the potential to reshape clinical practices and therapeutic outcomes in cancer and neurodegenerative diseases. In this dynamic and intriguing realm of cancer research, recent years witnessed a surge in attention towards understanding the intricate role of Mesenchymal Stem Cells (MSCs). These cells, capable of either suppressing or promoting tumors across diverse experimental models, have been a focal point in the exploration of exosome-based therapies. Exosomes released by MSCs have played a pivotal role, in unraveling the nuances of paracrine signaling and its profound impact on cancer development. Recent studies have revealed the complex nature of MSC-derived exosomes, showcasing both pro-tumor and anti-tumor effects. Despite their multifaceted involvement in tumor growth, these exosomes show significant promise in influencing both tumor development and chemosensitivity, acting as a pivotal factor that increases stem cells' potential for medicinal use. Endogenous MSCs, primarily originating from the bone marrow, exhibited a unique migratory response to damaged tissue sites. The genetic modification of stem cells, including MSCs, opened avenues for the precise delivery of therapeutic payloads in the milieu around the tumor (TME). Stem cell therapy offers groundbreaking potential for treating neurodegenerative and autoimmune disorders by regenerating damaged tissues and modulating immune responses. This approach aims to restore lost function and promote healing through targeted cellular interventions. In this review, we explored the molecular complexities of cancer and the potential for breakthroughs in personalized and targeted therapies. This analysis offers hope for transformative advancements in both cancer treatment and neurodegenerative disorders, highlighting the promise of precision medicine in addressing these challenging conditions.

Stem Cell Therapy for Liver Diseases: Current Perspectives.

Stem cell therapy offers a promising avenue for advanced liver disease cases as an alternative to liver transplantation. Clinical studies are underway to explore the potential of stem cells from various sources in treating different liver diseases. However, due to the variability among current studies, further validation is needed to ensure the safety and effectiveness of stem cell therapy. To establish a strong foundation for optimal stem cell therapy applications, selection of suitable stem cell sources, standardization of transplantation protocols, and patient criteria are vital. This review comprehensively examines existing literature on stem cell sources, transplantation methods, and patient selection. Additionally, we discuss novel strategies, including stem cell preconditioning, cell-free therapy, genetic modification of stem cells, and the use of liver organoids, addressing the limitations of current stem cell therapies. Nevertheless, these innovative approaches require further validation.

An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny.

SummaryThe genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics.

Gold nanoparticles for the in situ polymerization of near-infrared responsive hydrogels based on fibrin

Non-invasiveness and relative safety of photothermal therapy, which enables local hyperthermia of target tissues using a near infrared (NIR) laser, has attracted increasing interest. Due to their biocompatibility, amenability of synthesis and functionalization, gold nanoparticles have been investigated as therapeutic photothermal agents. In this work, hollow gold nanoparticles (HGNP) were coated with poly-l-lysine through the use of COOH-Poly(ethylene glycol)-SH as a covalent linker. The functionalized HGNP, which peak their surface plasmon resonance at 800 nm, can bind thrombin. Thrombin-conjugated HGNP conduct in situ fibrin polymerization, facilitating the process of generating photothermal matrices. Interestingly, the metallic core of thrombin-loaded HGNP fragmentates at physiological temperature. During polymerization process, matrices prepared with thrombin-loaded HGNP were loaded with genetically-modified stem cells that harbour a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation of resulting cell constructs in the presence of ligand successfully triggered transgene expression in vitro and in vivo. Statement of significanceCurrent technological development allows synthesis of gold nanoparticles (GNP) in a wide range of shapes and sizes, consistently and at scale. GNP, stable and easily functionalized, show low cytotoxicity and high biocompatibility. Allied to that, GNP present optoelectronic properties that have been exploited in a range of biomedical applications. Following a layer-by-layer functionalization approach, we prepared hollow GNP coated with a positively charged copolymer that enabled thrombin conjugation. The resulting nanomaterial efficiently catalyzed the formation of fibrin hydrogels which convert energy of the near infrared (NIR) into heat. The resulting NIR-responsive hydrogels can function as scaffolding for cells capable of controlled gene expression triggered by optical hyperthermia, thus allowing the deployment of therapeutic gene products in desired spatiotemporal frameworks.

Improved Transfection Efficiency and Metabolic Activity in Human Embryonic Stem Cell Using Non-Enzymatic Method.

Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in in vitro preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.

Evaluation of genotoxicity and mutagenic effects of vector/DNA nanocomplexes in transfected mesenchymal stem cells by flow cytometry

In recent years, there has been a great deal of interest in ex-vivo genetic modification of mesenchymal stem cells (MSCs) to meet various biomedical needs. Considering the self-renewal potential of MSCs, it is critically important to ensure that transfection vectors (gene carriers) do not induce genotoxicity because they could theoretically turn a single stem cell into a cancer-initiating cell. Unfortunately, there is currently no reliable, unbiased, and quantitative method to measure genotoxicity (micronuclei formation) of gene carriers directly in transfected MSCs. Consequently, it has not been possible to study the correlation of vectors’ physicochemical characteristics with their impact on stem cell genome stability. To address this deficiency, a flow cytometry-based method with a specialized gating protocol was developed that not only measures micronuclei formation, but also determines the mechanism of mutagenesis (i.e., clastogenic vs. aneugenic) of each vector in transfected MSCs. This gating protocol effectively eliminates all interfering signals associated with aggregated nanoparticles (viral and non-viral), exogenous DNA, and apoptotic/necrotic bodies from the micronuclei measurement process. The presented gating protocol for flow cytometry, which is provided as a template, enables investigators in academia, industry and regulatory bodies to rapidly and reliably evaluate the genosafety profiles of gene carriers. The findings of this study also indicate that highly positively charged lipid- and polymeric-based vectors can induce genotoxicity even without manifesting substantial somatic toxicity. Thus, extreme care must be taken before implanting ex-vivo-modified MSCs back into a patient’s body. Statement of SignificanceThere is a great interest in genetic modification of stem cells (SCs) by using vectors for various biomedical needs. Considering the self-renewal potential of SCs, it is essential to ensure that such vectors do not induce genetic aberrations (genotoxicity) because they could theoretically turn a single stem cell into a cancer-initiating cell. Unfortunately, there is currently no reliable method to measure genotoxicity of vectors directly in transfected SCs. To address this deficiency, a specialized flow cytometry-based method was developed that quantitatively analyzed genotoxicity and determined the mechanism of mutagenesis that occurred in transfected SCs during the transfection process. The developed technique will enable scientists to design safer vectors for genetic modification of stem cells.

Tube Formation Potential of BMSCs and USSCs in Response to HIF-1α Overexpression under Hypoxia

Despite numerous studies on therapeutic effects of mesenchymal stem cells on ischemic tissue regeneration, including angiogenesis, their mechanism of action remains ambiguous. Due to the scarce of investigations based on different stem cell sources with known inherent molecular differences, present study compare tube formation of Bone marrow Mesenchymal Stem Cells and Unrestricted Somatic Stem Cells with known reported different Hox gene expression profile in response to HIF-1α overexpression under hypoxia. This might shed light on some parameters for selection of more responsive source with improved therapeutic effects. Superior in vitro tube formation on Matrigel substratum has been observed by Unrestricted Somatic Stem Cells compared to Bone marrow Mesenchymal Stem Cells which might possibly be due to the discriminating molecular properties of stem cell sources. It may help choosing the appropriate stem cell type for a given therapeutic expectations and also suggests some potential targets for future genetic modification of stem cells.

Miro1 Enhances Mitochondria Transfer from Multipotent Mesenchymal Stem Cells (MMSC) to Neural Cells and Improves the Efficacy of Cell Recovery.

A recently discovered key role of reactive oxygen species (ROS) in mitochondrial traffic has opened a wide alley for studying the interactions between cells, including stem cells. Since its discovery in 2006, intercellular mitochondria transport has been intensively studied in different cellular models as a basis for cell therapy, since the potential of replacing malfunctioning organelles appears to be very promising. In this study, we explored the transfer of mitochondria from multipotent mesenchymal stem cells (MMSC) to neural cells and analyzed its efficacy under normal conditions and upon induction of mitochondrial damage. We found that mitochondria were transferred from the MMSC to astrocytes in a more efficient manner when the astrocytes were exposed to ischemic damage associated with elevated ROS levels. Such transport of mitochondria restored the bioenergetics of the recipient cells and stimulated their proliferation. The introduction of MMSC with overexpressed Miro1 in animals that had undergone an experimental stroke led to significantly improved recovery of neurological functions. Our data suggest that mitochondrial impairment in differentiated cells can be compensated by receiving healthy mitochondria from MMSC. We demonstrate a key role of Miro1, which promotes the mitochondrial transfer from MMSC and suggest that the genetic modification of stem cells can improve the therapies for the injured brain.

Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells

Vectors used for stem cell transfection must be non-genotoxic, in addition to possessing high efficiency, because they could potentially transform normal stem cells into cancer-initiating cells. The objective of this research was to bioengineer an efficient vector that can be used for genetic modification of stem cells without any negative somatic or genetic impact. Two types of multifunctional vectors, namely targeted and non-targeted were genetically engineered and purified from E. coli. The targeted vectors were designed to enter stem cells via overexpressed receptors. The non-targeted vectors were equipped with MPG and Pep1 cell penetrating peptides. A series of commercial synthetic non-viral vectors and an adenoviral vector were used as controls. All vectors were evaluated for their efficiency and impact on metabolic activity, cell membrane integrity, chromosomal aberrations (micronuclei formation), gene dysregulation, and differentiation ability of stem cells. The results of this study showed that the bioengineered vector utilizing VEGFR-1 receptors for cellular entry could transfect mesenchymal stem cells with high efficiency without inducing genotoxicity, negative impact on gene function, or ability to differentiate. Overall, the vectors that utilized receptors as ports for cellular entry (viral and non-viral) showed considerably better somato- and genosafety profiles in comparison to those that entered through electrostatic interaction with cellular membrane. The genetically engineered vector in this study demonstrated that it can be safely and efficiently used to genetically modify stem cells with potential applications in tissue engineering and cancer therapy.

Pim1 Kinase Overexpression Enhances ckit+ Cardiac Stem Cell Cardiac Repair Following Myocardial Infarction in Swine

BackgroundPim1 kinase plays an important role in cell division, survival, and commitment of precursor cells towards a myocardial lineage, and overexpression of Pim1 in ckit+ cardiac stem cells (CSCs) enhances their cardioreparative properties. ObjectivesThe authors sought to validate the effect of Pim1-modified CSCs in a translationally relevant large animal preclinical model of myocardial infarction (MI). MethodsHuman cardiac stem cells (hCSCs, n = 10), hckit+ CSCs overexpressing Pim1 (Pim1+; n = 9), or placebo (n = 10) were delivered by intramyocardial injection to immunosuppressed Yorkshire swine (n = 29) 2 weeks after MI. Cardiac magnetic resonance and pressure volume loops were obtained before and after cell administration. ResultsWhereas both hCSCs reduced MI size compared to placebo, Pim1+ cells produced a ∼3-fold greater decrease in scar mass at 8 weeks post-injection compared to hCSCs (−29.2 ± 2.7% vs. −8.4 ± 0.7%; p < 0.003). Pim1+ hCSCs also produced a 2-fold increase of viable mass compared to hCSCs at 8 weeks (113.7 ± 7.2% vs. 65.6 ± 6.8%; p <0.003), and a greater increase in regional contractility in both infarct and border zones (both p < 0.05). Both CSC types significantly increased ejection fraction at 4 weeks but this was only sustained in the Pim1+ group at 8 weeks compared to placebo. Both hCSC and Pim1+ hCSC treatment reduced afterload (p = 0.02 and p = 0.004, respectively). Mechanoenergetic recoupling was significantly greater in the Pim1+ hCSC group (p = 0.005). ConclusionsPim1 overexpression enhanced the effect of intramyocardial delivery of CSCs to infarcted porcine hearts. These findings provide a rationale for genetic modification of stem cells and consequent translation to clinical trials.

347. CRISPR/Cas9-Based Gene Correction of Arginase-Deficient Human Induced Pluripotent Stem Cells to Recover Enzyme Function

Urea cycle disorders (UCDs) are incurable genetic diseases that affect the body's ability to produce urea, leading to hyperammonemia due to a deficiency in any one of six enzymes in the cycle. For arginase deficiency, a mutation in the ARG1 gene, the final step of the cycle, results in hyperargininemia, developmental delays and disabilities, seizures, psychomotor function loss, and in serious cases, death. There is currently no completely effective treatment available. Advances in human induced pluripotent stem cell (hiPSC) research and genome-editing technologies have enabled the genetic modification of stem cells for potential cellular replacement therapies. In this study, we applied such technology to develop a stem cell-based approach for treating arginase deficiency applicable to all arginase deficient patients regardless of their mutation. Methods: Fibroblasts from three patients with arginase deficiency were obtained, defined for their mutation, and reprogrammed into hiPSCs. Selectable, full-length codon optimized human arginase cDNA (coARG) expression cassettes were then developed for site-specific integration into either the HPRT or albumin (ALB) locus. After confirming specificity by Sanger sequencing, genetically corrected hiPSCs were differentiated to hepatocyte-like cells. Results: Fibroblasts were reprogrammed into hiPSCs by applying a STEMCCA lentivirus-based method and were characterized for pluripotency by immunophenotyping for common stemness markers via ICC, alkaline phosphatase staining, and in vivo teratoma formation. Using a site-specific CRISPR/Cas9 nickase-mediated gene transferring system, we inserted coARG by two approaches. First, coARG was inserted into the HPRT locus under the control of constitutive hEF1α promoter (LEAPR); targeting HPRT allowed for positive clonal selection of successful on-target integration by 6-thioguanine treatment. Second, we inserted coARG into the ALB locus for expression under the endogenous ALB promoter (ALB-coARG) as ALB is highly expressed in human liver. After LEAPR and ALB-coARG modification and sequence confirmation, hiPSCs were differentiated to hepatocyte-like cells and characterized by immunophenotyping via ICC and RNA expression via RT-PCR for common hepatic markers; cells demonstrated more fetal-like characteristics. Moreover, LEAPR- and ALB-coARG-modified hepatocyte-like cells demonstrated 41% and 1% functional arginase activity recovery compared to human fetal liver, respectively. Discussion: In this study, we demonstrated the ability to genetically correct mutated ARG1 gene expression in hiPSCs derived from patients with hyperargininemia and restored arginase function in hiPSCs and hepatocyte derivatives by CRISPR/Cas9-based gene addition. As the LEAPR construct demonstrated marked coARG expression, recovery of arginase activity of ALB-ARG-modified hepatocyte-like cells was low; we expect significant arginase recovery after in vivo maturation of transplanted cells as ALB expression increases with hepatocyte maturation. Also, to demonstrate potential in vivo recovery of arginase deficiency pathogenesis, ongoing studies aim to transplant both cohorts of gene-corrected hepatocyte-like cells into an established arginase deficient immunosuppressed mouse model. Successful restoration of enzyme function in patient-specific hiPSCs and relevant cellular derivatives will highlight hiPSCs as a valuable tool in cell replacement therapies and advance applications of genetically modified hiPSCs to treat UCDs and other single enzyme liver deficiencies.

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

Concise Review: Patient-Derived Stem Cell Research for Monogenic Disorders.

Monogenic disorders (MGDs) are caused by a single gene mutation and have a serious impact on human health. At present, there are no effective therapeutic methods for MGDs. Stem cell techniques provide insights into potential treatments for MGDs. With the development of patient-derived stem cells, we can begin to progressively understand the molecular mechanism of MGDs and identify new drugs for MGD treatment. Using powerful genome editing tools, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regulatory interspaced short palindromic repeat/Cas9 system, MGD-associated gene mutations can be corrected in MGD stem cells in vitro and then transplanted into MGD animal models to assess their safety and therapeutic effects. Despite the continued challenges surrounding potential pluripotent stem cell tumorigenicity and concerns regarding the genetic modification of stem cells, the extensive clinical application of MGD patient-specific stem cells will be pursued through further advances in basic research in the MGD field. In this review, we will summarize the latest progress in research into the use of patient-derived stem cells for the potential treatment of MGDs and provide predictions regarding the direction of future investigations.

Engineering Stem Cells for Biomedical Applications.

Stem cells are characterized by a number of useful properties, including their ability to migrate, differentiate, and secrete a variety of therapeutic molecules such as immunomodulatory factors. As such, numerous pre-clinical and clinical studies have utilized stem cell-based therapies and demonstrated their tremendous potential for the treatment of various human diseases and disorders. Recently, efforts have focused on engineering stem cells in order to further enhance their innate abilities as well as to confer them with new functionalities, which can then be used in various biomedical applications. These engineered stem cells can take on a number of forms. For instance, engineered stem cells encompass the genetic modification of stem cells as well as the use of stem cells for gene delivery, nanoparticle loading and delivery, and even small molecule drug delivery. The present Review gives an in-depth account of the current status of engineered stem cells, including potential cell sources, the most common methods used to engineer stem cells, and the utilization of engineered stem cells in various biomedical applications, with a particular focus on tissue regeneration, the treatment of immunodeficiency diseases, and cancer.

Genome Editing in Stem Cells

Progress in the derivation, expansion and differentiation of human embryonic stem cells in vitro has led to the development of a valuable platform for studying disease as well as providing an inexhaustible resource for cell replacement therapies. Targeted modification of stem cells utilising synthetic nucleases provides a means to genetically alter the genome in an efficient, site-specific manner. Editing with this approach offers a possibility to improve disease modelling in both patient-derived induced pluripotent stem cells (iPSCs) and embryonic and adult stem cells. It is possible that in the future, patients with monogenic diseases could be treated or cured by transplantation of autologous cells that have been altered to have the corrected genetic sequence. Currently, genetic modification of stem cells in vitro is most often achieved using siRNA or viral transduction; however, this can result in only transient modulation or raise the possibility of insertional mutagenesis, respectively. Targeted genome editing using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and more recently clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated protein 9 (Cas9) have been developed to overcome the drawbacks of gene transfer. In this review, we mechanistically outline these technologies before focusing on their expanding utility within the biomedical sciences.

Genistein promotes endothelial colony-forming cell (ECFC) bioactivities and cardiac regeneration in myocardial infarction.

Although stem cell-mediated treatment of ischemic diseases offers significant therapeutic promise, the limitation in the therapeutic efficacy of transplanted stem cells in vivo because of poor engraftment remains a challenge. Several strategies aimed at improving survival and engraftment of stem cells in the ischemic myocardium have been developed, such as cell transplantation in combination with growth factor delivery, genetic modification of stem cells, and/or cell therapy using scaffolds. To improve therapeutic efficacy, we investigated the effects of genistein on the engraftment of transplanted ECFCs in an acute myocardial ischemia model. Results: We found that genistein treatment enhanced ECFCs' migration and proliferation, which was accompanied by increases in the expression of ILK, α-parvin, F-actin, and phospholylation of ERK 1/2 signaling. Transplantation of genistein-stimulates ECFCs (GS-ECFCs) into myocardial ischemic sites in vivo induced cellular proliferation and secretion of angiogenic cytokines at the ischemic sites and thereby enhanced neovascularization and decreased myocardial fibrosis as well as improved cardiac function, as shown by echocardiography. Taken together, these data suggest that pretreatment of ECFCs with genistein prior to transplantation can improve the regenerative potential in ischemic tissues, providing a novel strategy in adult stem cell therapy for ischemic diseases.

Application of Biologics in the Treatment of the Rotator Cuff, Meniscus, Cartilage, and Osteoarthritis

Advances in our knowledge of cell signaling and biology have led to the development of products that may guide the healing/regenerative process. Therapies are emerging that involve growth factors, blood-derived products, marrow-derived products, and stem cells. Animal studies suggest that genetic modification of stem cells will be necessary; studies of cartilage and meniscus regeneration indicate that immature cells are effective and that scaffolds are not always necessary. Current preclinical animal and clinical human data and regulatory requirements are important to understand in light of public interest in these products.

Gene Delivery Techniques for Adult Stem Cell-Based Regenerative Therapy

Over the past decade, stem cells have been considered to be a promising resource to cure and regenerate damaged or diseased tissues with research extending from basic studies to clinical application. Furthermore, genetically modified stem cells have the potential to reduce tumorigenic risks and achieve safe tissue formation. Recent advances in genetic modification of stem cells have rendered these cells more accessible and stable. The successful genetic modification of stem cells relies heavily on designing vector systems, either viral or nonviral vectors, which can efficiently deliver therapeutic genes to the cells with minimum toxicity. Currently, viral vectors showing high transfection efficiencies still raise safety issues, whereas safer nonviral vectors exhibit extremely poor transfection in stem cells. Here, we attempt to review and discuss the main factors raising concern in previous reports, and devise strategies to solve the issues in gene delivery systems for successful stem cell-targeting regenerative therapy.

Innovative Strategy for MicroRNA Delivery in Human Mesenchymal Stem Cells via Magnetic Nanoparticles

Bone marrow derived human mesenchymal stem cells (hMSCs) show promising potential in regeneration of defective tissue. Recently, gene silencing strategies using microRNAs (miR) emerged with the aim to expand the therapeutic potential of hMSCs. However, researchers are still searching for effective miR delivery methods for clinical applications. Therefore, we aimed to develop a technique to efficiently deliver miR into hMSCs with the help of a magnetic non-viral vector based on cationic polymer polyethylenimine (PEI) bound to iron oxide magnetic nanoparticles (MNP). We tested different magnetic complex compositions and determined uptake efficiency and cytotoxicity by flow cytometry. Additionally, we monitored the release, processing and functionality of delivered miR-335 with confocal laser scanning microscopy, real-time PCR and live cell imaging, respectively. On this basis, we established parameters for construction of magnetic non-viral vectors with optimized uptake efficiency (~75%) and moderate cytotoxicity in hMSCs. Furthermore, we observed a better transfection performance of magnetic complexes compared to PEI complexes 72 h after transfection. We conclude that MNP-mediated transfection provides a long term effect beneficial for successful genetic modification of stem cells. Hence, our findings may become of great importance for future in vivo applications.

Going 3D – Cell Culture Approaches for Stem Cell Research and Therapy

Current stem cell research greatly depends on suitable in vitro-cultivation approaches, enabling expansion, differentiation, cryopreservation or genetic modification of stem cells outside the organism. Particularly regarding neurodegenerative diseases, regeneration of complex injuries or cancer, this already great field of applications is even broadened by in vitro-culture approaches for stem cell-based therapy. Here, 2D-concepts of cultivation focus on cell differentiation or short-time expansion for further transplantation as well as the elucidation of molecular mechanisms of a certain disease for drug targeting. However, latest studies suggest potential beneficial effects of 3D cultivation strategies. In contrast to 2D-culture, the conditions of the endogenous stem cell niche are mirrored more closely, leading to improved cellular viability and differentiation behavior. The use of 3D-cultivated stem cell-products may provide the advantages of direct transplantation, enhanced graft adherence and higher cellular loading densities within the transplant. Thus, together with the increased differentiation potential of stem cells under such conditions, 3D-culture may provide a powerful tool for regenerative medicine. Here, we summarize current 3D-cultivation approaches for adult, embryonic and cancer stem cells, highlighting their potential scientific and clinical impacts.