Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.     

Electrical Stimulation of Human Mesenchymal Stem Cells on Conductive Substrates Promotes Neural Priming

Electrical stimulation (ES) within a conductive scaffold is potentially beneficial in encouraging the differentiation of stem cells toward a neuronal phenotype. To improve stem cell-based regenerative therapies, it is essential to use electroconductive scaffolds with appropriate stiffnesses to regulate the amount and location of ES delivery. Herein, biodegradable electroconductive substrates with different stiffnesses are fabricated from chitosan-grafted-polyaniline (CS-g-PANI) copolymers. Human mesenchymal stem cells (hMSCs) cultured on soft conductive scaffolds show a morphological change with significant filopodial elongation after electrically stimulated culture along with upregulation of neuronal markers and downregulation of glial markers. Compared to stiff conductive scaffolds and non-conductive CS scaffolds, soft conductive CS-g-PANI scaffolds promote increased expression of microtubule-associated protein 2 (MAP2) and neurofilament heavy chain (NF-H) after application of ES. At the same time, there is a decrease in the expression of the glial markers glial fibrillary acidic protein (GFAP) and vimentin after ES. Furthermore, the elevation of intracellular calcium [Ca²⁺] during spontaneous, cell-generated Ca²⁺ transients further suggests that electric field stimulation of hMSCs cultured on conductive substrates can promote a neural-like phenotype. The findings suggest that the combination of the soft conductive CS-g-PANI substrate and ES is a promising new tool for enhancing neuronal tissue engineering outcomes.     

Generation and characterization of integration-free induced pluripotent stem cells from patients with autoimmune disease

Autoimmune diseases (AIDs), a heterogeneous group of immune-mediated disorders, are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs, the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However, stem cell transplantation therapy has limitations, including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology, which allows the generation of patient-specific pluripotent stem cells, could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS), Sjögren''s syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally, all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS, SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs, including AS, SS and SLE.     

Ile‐Lys‐Val‐ala‐Val (IKVAV) peptide for neuronal tissue engineering

Despite the great advances in microsurgery, some neural injuries cannot be treated surgically. Stem cell therapy is a potential approach for treating neuroinjuries and neurodegenerative disease. Researchers have developed various bioactive scaffolds for tissue engineering, exhibiting enhanced cell viability, attachment, migration, neurite elongation, and neuronal differentiation, with the aim of developing functional tissue grafts that can be incorporated in vivo. Facilitating the appropriate interactions between the cells and extracellular matrix is crucial in scaffold design. Modification of scaffolds with biofunctional motifs such as growth factors, drugs, or peptides can improve this interaction. In this review, we focus on the laminin‐derived Ile‐Lys‐Val‐Ala‐Val peptide as a biofunctional epitope for neuronal tissue engineering. Inclusion of this bioactive peptide within a scaffold is known to enhance cell adhesion as well as neuronal differentiation in both 2‐dimensional and 3‐dimensional environments. The in vivo application of this peptide is also briefly described.     

3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body

This paper proposes a microfluidic device for the on-chip differentiation of an embryoid body (EB) formed in a microwell via 3-dimensional cultures of mouse embryonic carcinoma (EC) cells. The device adjusted the size of the EB by fluid volume, differentiated the EB by chemical treatment, and evaluated its effects in EC cells by on-chip immunostaining. A microfluidic resistance network was designed to control the size of the embryoid body. The duration time and flow rate into each microwell regulated the initial number of trapped cells in order to adjust the size of the EB. The docked cells were aggregated and formed a spherical EB on the non-adherent surface of the culture chip for 3 days. The EC cells in the EB were then differentiated into diverse cell lineages without attachment for an additional 4 days; meanwhile, retinoic acid (RA) was applied without serum to direct the cells into early neuronal lineage. On-chip immunostaining of the EB in the microwell with a neuronal marker was conducted to assess the differentiation-inducing ability of RA. The effect of RA on neuronal differentiation was analyzed with confocal microscopic images of the TuJ1 marker. The RA-treated cells expressed more neuronal markers and appeared as mature neuronal cells with long neurites. The fluorescence intensity of the TuJ1 in the RA-treated EB was twice that observed in the non-treated EB on day 5. It was demonstrated that the pre-screening of inducing chemicals on the early neuronal differentiation of EC cells in a single microfluidic chip was indeed feasible. This chip is expected to constitute a useful tool for assessing the early differentiation of ES cells without attachment, and is also expected to prove useful as an anti-cancer drug test platform for the cytotoxicity assay with cellular spheroids.     

Enhancing endothelial differentiation of human mesenchymal stem cells by culture on a nanofibrous polycaprolactone/(poly-glycerol sebacate)/gelatin scaffold

Cardiovascular diseases have always been one of the main causes of death worldwide and eventually one of the major medical concerns. Tissue engineering is promising strategies of treating cardiovascular, which can be an effective approach with the design of appropriate scaffold. In this study, to develop engineering basement membrane for endothelial differentiation with good cell attachment, we produced polycaprolactone (PCL)/poly (glycerol sebacate) (PGS)/gelatin nanofibrous scaffold via electrospinning. Attenuated total reflectance-Fourier transform infrared and the proton nuclear magnetic resonance results confirmed the chemical structure of synthesized PGS. Scanning electron microscope images of the electrospun scaffold revealed that the nanofibers are smooth, continues and uniform. Moreover, due to the presence of hydrophilic functional groups in the scaffold, the contact angle is in the appropriate range for cell adhesion especially endothelial cells. The elastic modulus and ultimate tensile stress of electrospun scaffold were calculated 1.32 ± 0.27 MPa and 1.23 ± 0.18 MPa respectively. Quantitative polymerase chain reaction was performed for evaluation of endothelial differentiation of mesenchymal stem cells cultured on standard plate and fibrous scaffold under chemical stimulation with growth factor. Specific endothelial gene expression results postulated that our modified scaffold could support and significantly promote endothelial differentiation of MSCs.     

Biomimetic Mineralized Fiber Bundle-Inspired Scaffolding Surface on Polyetheretherketone Implants Promotes Osseointegration

The stress shielding effect caused by traditional metal implants is circumvented by using polyetheretherketone (PEEK), due to its excellent mechanical properties; however, the biologically inert nature of PEEK limits its application. Endowing PEEK with biological activity to promote osseointegration would increase its applicability for bone replacement implants. A biomimetic study is performed, inspired by mineralized collagen fiber bundles that contact bone marrow mesenchymal stem cells (BMMSCs) on the native trabecular bone surface. The PEEK surface (P) is first sulfonated with sulfuric acid to form a porous network structure (sP). The surface is then encapsulated with amorphous hydroxyapatite (HA) by magnetron sputtering to form a biomimetic scaffold that resembles mineralized collagen fiber bundles (sPHA). Amorphous HA simulates the composition of osteogenic regions in vivo and exhibits strong biological activity. In vitro results show that more favorable cell adhesion and osteogenic differentiation can be attained with the novelsurface of sPHA than with SP. The results of in vivo experiments show that sPHA exhibits osteoinductive and osteoconductive activity and facilitates bone formation and osseointegration. Therefore, the surface modification strategy can significantly improve the biological activity of PEEK, facilitate effective osseointegration, and inspire further bionic modification of other inert polymers similar to PEEK.     

Restraint of the Differentiation of Mesenchymal Stem Cells by a Nonfouling Zwitterionic Hydrogel

The success of human mesenchymal stem cell (hMSC) therapies is largely dependent on the ability to maintain the multipotency of cells and control their differentiation. External biochemical and biophysical cues can readily trigger hMSCs to spontaneously differentiate, thus resulting in a rapid decrease in the multipotent cell population and compromising their regenerative capacity. Herein, we demonstrate that nonfouling hydrogels composed of pure poly(carboxybetaine) (PCB) enable hMSCs to retain their stem‐cell phenotype and multipotency, independent of differentiation‐promoting media, cytoskeletal‐manipulation agents, and the stiffness of the hydrogel matrix. Moreover, encapsulated hMSCs can be specifically induced to differentiate down osteogenic or adipogenic pathways by controlling the content of fouling moieties in the PCB hydrogel. This study examines the critical role of nonspecific interactions in stem‐cell differentiation and highlights the importance of materials chemistry in maintaining stem‐cell multipotency and controlling differentiation.     

High Content Screening for Compounds that Induce Early Stages of Human Embryonic Stem Cell Differentiation

Embryonic stem cells, due to their self-renewal and pluripotency properties, can be used to repair damaged tissues and as an unlimited source of differentiated cells. Although stem cells represent an important opportunity for cell based therapy and small molecules screening (in the context of drug or target discovery) many drawbacks are still preventing their widespread use. One of the most significant limitations is related to the complexity, as well as the reliability, of current protocols driving stem cells into any homogeneously differentiated cellular population. In this respect there is a strong demand for molecular agents promoting differentiation and thereby enabling robust, efficient and safe production of differentiated cells. In order to identify novel molecules that enhance early stages of differentiation, we developed an image based high content screening (HCS) approach using human embryonic stem cells (hESC). In our approach, we took advantage of custom image mining software specifically adapted for the selection of stem cell differentiation agents and the rejection of false positive hits. As a proof of concept ～3500 small molecules originating from commercial libraries were screened and a number of molecules of interests were identified. These molecules show stem cell differentiation properties comparable to the phenotypic signature obtained with the reference compound retinoic acid.     

Enhanced differentiation of embryonic and neural stem cells to neuronal fates on laminin peptides doped polypyrrole

PPy is a conducting polymer material that has been widely investigated for biomedical applications. hESCs and adult rNSCs were grown on four PPy surfaces doped with PSS or peptide from laminin (p20, p31, and a mixture of p20 and p31) respectively. After 7 d, both PPy/p20 and PPy/p31 promoted neuroectoderm formation from hESCs. After 14 d of culture, surfaces containing p20 showed the highest percentage of neuronal differentiation from hESC, while the PPy/p31 surface showed better cell attachment and spreading. In rNSCs cultures, a higher percentage of neurons were found on the PPy/p20 surface than other surfaces at 7 and 14 d. For differentiated neurons, p20 promoted both the primary and total neurite outgrowth. Longer primary neurites were found on p20-containing surfaces and a longer total neurite length was found on PPy/p20 surface. These results demonstrated that, by doping PPy with different bioactive peptides, differentiation of stem cells seeded at different stages of development is affected.     

Using the chorions of fertilized zebrafish eggs as a biomaterial for the attachment and differentiation of mouse stem cells

The development of proper biomaterials is critical for the success of cell therapy and modern tissue engineering. Here, we extruded the yolk and remaining inner mass from fertilized zebrafish eggs and used the resulting chorions as a biomaterial for the differentiation and attachment of mouse P19 embryonic carcinoma (EC) cells. Cells inserted into the chorion showed the spontaneous formation of embryoid body due to the repulsive cell adhesion of the chorion and differentiated specifically into neural cells and cardiomyocytes. In contrast, dissolved chorion extracellular matrix (ECM) conferred enhanced cell attachment on it, suggesting that a unique property of the zebrafish chorion with nanoporous structure appears to be responsible for the simple and controllable embryoid formation for stem cell differentiation. These results indicate that chorions from fertilized zebrafish eggs may be used as an extracellular matrix alternative and applied for stem cell differentiation to specific cell lineages.     

Three-dimensional extracellular matrix-mediated neural stem cell differentiation in a microfluidic device

Here, we report a unique method to quantify the effects of in vivo-like extracellular matrix (ECM) for guiding differentiation of neural stem cells (NSCs) in three-dimensional (3D) microenvironments using quantitative real-time polymerase chain reaction (qRT-PCR). We successfully monitored and quantified differentiation of NSCs in small volume ECMs and found that differentiation of NSCs, especially those differentiating towards neuronal and oligodendrocytic lineages, is significantly enhanced by 3D microenvironments reconstituted in the microfluidic channels.     

The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells

Icariin had been reported as a potential agent for osteogenesis, but the dose-effect relationship needed further research to realize the clinical application of icariin. We isolated and purified human bone mesenchymal stem cells (hBMSCs) and stimulated them with different concentrations of icariin. The cytotoxicity of icariin was evaluated by the methylthiazolytetrazolium (MTT) assay method. The proliferation and osteogenic differentiation of such hBMSCs were investigated for different concentrations of icariin. We found that icariin had a dose-dependent effect on the proliferation and osteogenic differentiation of hBMSCs in a suitable concentration range from 10(-9) M to 10(-6) M, but at concentrations above 10(-5) M, the cytotoxicity limited its use. The extremely low cost of icariin and its high abundance make it appealing for bone regeneration.     

Differentiation of individual human mesenchymal stem cells probed by FTIR microscopic imaging

Objective of this study is the novel application of Fourier transform infrared (FTIR) microscopic imaging to identify the differentiation state of individual human mesenchymal stem cells with or without osteogenic stimulation. IR spectra of several hundred single cells with lateral resolution of 5-10 microm were recorded using a FTIR imaging spectrometer coupled to a microscope with a focal plane array detector. A classification model based on linear discriminant analysis was trained to distinguish four cell types by their IR spectroscopic fingerprint. Without stimulation two cell types dominated, showing low or high levels of glycogen accumulation at the cell periphery. After stimulation, the protein composition in the cells changed and some cells started expressing calcium phosphate salts such as octacalciumphosphate, a precursor of the bone constituent hydroxyapatite. Few cells were identified which remained in their non-stimulated state. This study demonstrated for the first time that FTIR microscopic imaging can probe stem cell differentiation at the single cell level rapidly, non-destructively and with minimal preparation.     

Recruitment of endogenous stem cells for tissue repair

The traditional concept of stem cell therapy envisions the isolation of stem cells from patients, propagation and differentiation in vitro, and subsequent re-injection of autologous cells into the patient. There are many problems associated with this paradigm, particularly during the in vitro manipulation process and the delivery and local retention of re-injected cells. An alternative paradigm that could be easier, safer, and more efficient, would involve attracting endogenous stem cells and precursor cells to the defect site for new tissue regeneration. Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin, exerts a strong chemoattractive effect on mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and induces migration of MSCs in vitro. However, HGF undergoes rapid proteolysis in vivo, which results in a very short lifetime of the bioactive cytokine. To maintain the therapeutic level of HGF at the defect site necessary for endogenous stem cell recruitment, sustained, long-term, and localized delivery of HGF is required. Thiol-modified glycosaminoglycans hyaluronan (HA) and heparin (HP), combined with modified gelatin (Gtn), have been crosslinked with poly(ethylene glycol) diacrylate (PEGDA) to afford semisynthetic ECM-like (sECM) hydrogels that can both provide controlled growth factor release and permit cell infiltration and proliferation. Herein we compare the use of different sECM compositions for controlled release of HGF and concomitant recruitment of human bone marrow MSCs into the scaffold in vitro. [Figure: see text].     

Evaluation of 3D Printed Gelatin‐Based Scaffolds with Varying Pore Size for MSC‐Based Adipose Tissue Engineering

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion‐based 3D printing is used to fabricate photo‐crosslinkable gelatin‐based scaffolds with pore sizes in the range of 200–600 µm. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC‐based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.     

Tantalum nanoparticles enhance the osteoinductivity of multiscale composites based on poly(lactide-co-glycolide) electrospun fibers embedded in a gelatin hydrogel

Bioresorbable polymeric materials have risen great interest as implants for bone tissue regeneration, since they show substantial advantages with respect to conventional metal devices, including biodegradability, flexibility, and the possibility to be easily modified to introduce specific functionalities. In the present work, an innovative nanocomposite scaffold, properly designed to show biomimetic and osteoinductive properties for potential application in bone tissue engineering, was developed. The scaffold is characterized by a multi-layer structure, completely different with respect to the so far employed polymeric implants, consisting in a poly(d,l-lactide-co-glycolide)/polyethylene glycol electrospun nanofibrous mat sandwiched between two hydrogel gelatin layers enriched with tantalum nanoparticles (NPs). The composition of the electrospun fibers, containing 10 wt% of polyethylene glycol, was selected to ensure a proper integration of the fibers in the gel phase, essential to endow the composite with flexibility and to prevent delamination between the layers. The scaffold maintained its structural integrity after six weeks of soaking in physiological solutions, albeit the gelatin phase was partially released. The combined use of gelatin, bioresorbable electrospun fibers and tantalum NPs endows the final device with biomimetic and osteoinductive properties. Indeed, results of the in vitro tests demonstrate that the obtained scaffolds clearly represent a favorable milieu for normal human bone-marrow derived mesenchymal stem cells viability and osteoblastic differentiation; moreover, inclusion of tantalum NPs in the scaffold improves cell performance with particular regard to early and late markers of osteoblastic differentiation.     

丝素改性聚乳酸-羟基乙酸共聚物多孔微球作为牙龈间充质干细胞递送载体的研究

利用复乳-溶剂挥发法合成适合细胞三维培养的聚乳酸-羟基乙酸共聚物(PLGA)多孔微球, 并对其表面进行丝素改性, 利用扫描电子显微镜、 能谱、 红外光谱和X射线衍射等对改性前后PLGA多孔微球的理化特性进行表征. 原代培养人牙龈间充质干细胞并进行成骨(茜素红染色)成脂(油红O染色)分化鉴定. 通过负压混悬法将牙龈干细胞负载于丝素改性的PLGA多孔微球上进行5-乙炔基-2'-脱氧尿嘧啶核苷(EdU)细胞增殖及成骨分化研究. 结果表明, 原代培养的牙龈干细胞具有多向分化潜能, 负载在丝素改性的PLGA多孔微球上的细胞有利于细胞增殖. 丝素改性的PLGA多孔微球是良好的细胞递送载体, 为进一步修复牙槽骨缺损提供了科学依据.