Lineage-specific Differentiation Of Stem Cells Research Articles

Discreteness of cell–surface contacts affects spatio-temporal dynamics, adhesion, and proliferation of mouse embryonic stem cells

The self-renewal and lineage-specific differentiation of stem cells are regulated by interactions with their microenvironments, called stem cell niche. Stem cells receive both biochemical and biophysical cues from their niche, which leads to the activation of signaling pathways, resulting in the modulation of gene expressions to guide their fate. Most of previous studies are focused on the effect of substrate stiffness using hydrogels with different Young’s moduli, and information is lacking on the effect of the discreteness of cell–substrate contacts on stem cells. Using mouse pluripotent, embryonic stem cells (mESCs) as the model system for early development, we quantitatively investigated the migration, dynamic deformation, and adhesion of mESCs on sparse and dense gelatin nanofibers deposited on glass surfaces, with a continuous layer of gelatin coated on glass substrates as the control. After confirming the maintenance of pluripotency on all the surfaces throughout the experiments, the centroid trajectories were monitored using timelapse imaging. The mean square displacement analysis indicated that both the diffusion coefficient and exponent were largest on sparse nanofibers, while the diffusion coefficient of mESCs on dense nanofibers was comparable to that on the control. Moreover, power spectral analysis of the shape deformation in the Fourier mode indicated that mESCs predominantly underwent elliptic deformation (mode 2), with the largest energy dissipation on sparse nanofibers. These data suggest that mESCs can deform and move on sparse nanofibers owing to the discrete cell–surface contact points. Intriguingly, using a self-developed technique based on laser-induced shock waves, a distinctly larger critical pressure was required to detach cells from nanofibers than from continuous gelatin. This finding suggests that the continuous but weak cell-substrate contacts suppress the deformation-driven mESC migration. As one of the key biological functions of stem cells, the proliferation rate of mESCs on these surfaces was determined. Although the observed difference was not statistically significant, the highest proliferation rate was observed on nanofibers, suggesting that the discreteness of cell–surface contacts can be used to regulate not only spatio-temporal dynamics but also the biological function of pluripotent stem cells.

Exosomes as Biochemistry Tools for Stem Cell Differentiation: A Novel Cell-Based Treatment for Diseases

: For tissue engineering and clinical translation strategies, it is essential to have a reliable and safe lineage-specific differentiation of stem cells. To deal with several problems caused by growth factor delivery systems and growth factors, exosomes have been used as biomimetic tools to trigger the differentiation of stem cells. It is believed that cell type-specific exosomes can induce lineage-specific differentiation of stem cells. Exosomes trigger cell viability, cell proliferation and differentiation, embryonic implantation, and migration. They have been used successfully in regenerative medicine, such as liver fibrosis, renal diseases, cardiac ischemia, stroke, and skin injuries. The findings highlighted the necessity to take into account the condition and source of exosome donor cells before selecting them for therapeutic use.

MiR-342-5p inhibits odonto/osteogenic differentiation of human dental pulp stem cells via targeting Wnt7b.

Human dental pulp stem cells (hDPSCs) constitute a promising source of stem cells in tissue engineering. However, the molecular mechanism of differentiation in hDPSCs remains largely unclear. MicroRNAs (miRNAs) play crucial roles in lineage-specific differentiation of stem cells. The present study investigated the function of miRNA-342-5p in the odonto/osteogenic differentiation of hDPSCs. The miRNA array profiling and quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) revealed the expression of miR-342-5p during odonto/osteogenic differentiation of hDPSCs. hDPSCs were treated with miR-342-5p mimic and inhibitor to investigate the regulatory roles of miR-342-5p in the differentiation of hDPSCs. Moreover, miR-342-5p inhibitor and small interference RNA (siRNA) targeting Wnt7b were applied to explore the regulatory mechanism of miR-342-5p. Downregulated miR-342-5p was observed during odonto/osteogenic differentiation of hDPSCs. The overexpression of miR-342-5p inhibited the odonto/osteogenic potential of DPSCs, as indicated by low levels of alkaline phosphatase activity, calcium deposition formation, and odonto/osteogenic differentiation markers, whereas silencing of miR-342-5p exhibited the opposite effect. When co-treated with siRNA targeting Wnt7b and miR-342-5p inhibitor in hDPSCs, the odonto/osteogenic potential and activation of Wnt7b/β-catenin pathway were attenuated. This study showed that miR-342-5p inhibits the odonto/osteogenic differentiation of hDPSCs by interfering with Wnt/β-catenin signaling via targeting Wnt7b.

Mechanical stretch promotes antioxidant responses and cardiomyogenic differentiation in P19 cells

Accumulating evidence has suggested that mechanical stimuli play a crucial role in regulating the lineage-specific differentiation of stem cells through fine-tuning redox balance. We aimed to investigate the effects of cyclic tensile strain (CTS) on the expression of antioxidant enzymes and cardiac-specific genes in P19 cells, a widely characterized tool for cardiac differentiation research. A stretching device was applied to generate different magnitude and duration of cyclic strains on P19 cells. The messenger RNA and protein levels of targeted genes were determined by real-time polymerase chain reaction and Western blot assays, respectively. Proper magnitude and duration of cognitivestimulationtherapy (CST) stimulation substantially enhanced the expression of both antioxidant enzymes and cardiac-specific genes in P19 cells. Sirtuin 1 (SIRT1) played an essential role in the CTS-induced cardiomyogenic differentiation of P19, as evidenced by changes in the expression of antioxidant enzymes and cardiac-specific genes. Mechanical loading promoted the cardiomyogenic differentiation of P19 cells. SIRT1 was involved in CST-mediated P19 differentiation, implying that SIRT1 might serve as an important target for developing methods to promote cardiomyogenic differentiation of stem cells.

Enhanced growth and myogenic differentiation of spheroid-derived C2C12 cells.

Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs is significantly higher than the PCs in a time-dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.

Human cardiomyocyte-derived exosomes induce cardiac gene expressions in mesenchymal stromal cells within 3D hyaluronic acid hydrogels and in dose-dependent manner

Accomplishing a reliable lineage-specific differentiation of stem cells is vital in tissue engineering applications, however, this need remained unmet. Extracellular nanovesicles (particularly exosomes) have previously been shown to have this potential owing to their rich biochemical content including proteins, nucleic acids and metabolites. In this work, the potential of human cardiomyocytes-derived exosomes to induce in vitro cardiac gene expressions in human mesenchymal stem cells (hMSCs) was evaluated. Cardiac exosomes (CExo) were integrated with hyaluronic acid (HA) hydrogel, which was functionalized with tyramine (HA-Tyr) to enable the development of 3D (three dimensional), robust and bioactive hybrid cell culture construct through oxidative coupling. In HA-Tyr/CExo 3D hybrid hydrogels, hMSCs exhibited good viability and proliferation behaviours. Real time quantitative polymerase chain reaction (RT-qPCR) results demonstrated that cells incubated within HA-Tyr/CExo expressed early cardiac progenitor cell markers (GATA4, Nkx2.5 and Tbx5), but not cTnT, which is expressed in the late stages of cardiac differentiation and development. The expressions of cardiac genes were remarkably increased with increasing CExo concentration, signifying a dose-dependent induction of hMSCs. This report, to some extent, explains the potential of tissue-specific exosomes to induce lineage-specific differentiation. However, the strategy requires further mechanistic explanations so that it can be utilized in translational medicine.

Recent Progress in Engineering Mesenchymal Stem Cell Differentiation.

Due to the ability to differentiate into variety of cell types, mesenchymal stem cells (MSCs) hold promise as source in cell-based therapy for treating injured tissue and degenerative diseases. The potential use of MSCs to replace or repair damaged tissues may depend on the efficient differentiation protocols to derive specialized cells without any negative side effects. Identification of appropriate cues that support the lineage-specific differentiation of stem cells is critical for tissue healing and cellular therapy. Recently, a number of stimuli have been utilized to direct the differentiation of stem cells. Biochemical stimuli such as small molecule, growth factor and miRNA have been traditionally used to regulate the fate of stem cells. In recent years, many studies have reported that biophysical stimuli including cyclic mechanical strain, fluid shear stress, microgravity, electrical stimulation, matrix stiffness and topography can also be sensed by stem cells through mechanical receptors, thus affecting the stem cell behaviors including their differentiation potential. In this paper, we review all the most recent literature on the application of biochemical and biophysical cues on regulating MSC differentiation. An extensive literature search was done using electronic database (Medline/Pubmed). Although there are still some challenges that need to be taken into consideration before translating these methods into clinics, biochemical and biophysical stimulation appears to be an attractive method to manipulate the lineage commitment of MSCs.

Spatially controlled functional group grafting of silk films to induce osteogenic and chondrogenic differentiation of human mesenchymal stem cells

For tissue engineering, fabrication of appropriate biomaterials, which not only support cellular attachment and proliferation but also direct lineage-specific differentiation of stem cells is crucial. It is widely reported in literature that biomaterial surface chemistry modulates human mesenchymal stem cell (hMSC) differentiation in a lineage dependent manner. In recent years, natural materials such as silk have been used for hMSC culture because of its superior mechanical strength, biocompatibility, and biodegradability. We report here a simple strategy for differentiating hMSCs into two different lineages on the same surface. We have grafted functional groups such as acrylic acid and phosphates to guide the differentiation of hMSCs into chondrocytes and osteocytes respectively. Unlike other strategies, our strategy does not require growth factors and other added signals in the media and is initiated due to the difference of functional groups present on the surface. We believe this study will foster the development of effective silk based tissue engineered constructs.

Amyloids Are Novel Cell-Adhesive Matrices.

Amyloids are highly ordered peptide/protein aggregates traditionally associated with multiple human diseases including neurodegenerative disorders. However, recent studies suggest that amyloids can also perform several biological functions in organisms varying from bacteria to mammals. In many lower organisms, amyloid fibrils function as adhesives due to their unique surface topography. Recently, amyloid fibrils have been shown to support attachment and spreading of mammalian cells by interacting with the cell membrane and by cell adhesion machinery activation. Moreover, similar to cellular responses on natural extracellular matrices (ECMs), mammalian cells on amyloid surfaces also use integrin machinery for spreading, migration, and differentiation. This has led to the development of biocompatible and implantable amyloid-based hydrogels that could induce lineage-specific differentiation of stem cells. In this chapter, based on adhesion of both lower organisms and mammalian cells on amyloid nanofibrils, we posit that amyloids could have functioned as a primitive extracellular matrix in primordial earth.

Exosomes as biomimetic tools for stem cell differentiation: Applications in dental pulp tissue regeneration

Achieving and maintaining safe and reliable lineage specific differentiation of stem cells is important for clinical translation of tissue engineering strategies. In an effort to circumvent the multitude of problems arising from the usage of growth factors and growth factor delivery systems, we have explored the use of exosomes as biomimetic tools to induce stem cell differentiation. Working on the hypothesis that cell-type specific exosomes can trigger lineage-specific differentiation of stem cells, we have evaluated the potential of exosomes derived from dental pulp cells cultured on under growth and odontogenic differentiation conditions to induce odontogenic differentiation of naïve human dental pulp stem cells (DPSCs) and human bone marrow derived stromal cells (HMSCs) invitro and invivo. Results indicate that the exosomes can bind to matrix proteins such as type I collagen and fibronectin enabling them to be tethered to biomaterials. The exosomes are endocytosed by both DPSCs and HMSCs in a dose-dependent and saturable manner via the caveolar endocytic mechanism and trigger the P38 mitogen activated protein kinase (MAPK) pathway. In addition, the exosomes also trigger the increased expression of genes required for odontogenic differentiation. When tested invivo in a tooth root slice model with DPSCs, the exosomes triggered regeneration of dental pulp-like tissue. However, our results indicate that exosomes isolated under odontogenic conditions are better inducers of stem cell differentiation and tissue regeneration. Overall, our results highlight the potential exosomes as biomimetic tools to induce lineage specific differentiation of stem cells. Our results also show the importance of considering the source and state of exosome donor cells before a choice is made for therapeutic applications.

Roles of Mechanical Force and Mechanotransduction in Fate of Stem Cells

Stem cells are undifferentiated progenitor cells that are accepted as a source of seed cells for therapeutic applications in tissue regeneration and as a model to investigate the lineage-specific differentiation of stem cells. Outstanding research in recent years has shed light on the role of micro-environment of stem cells in the proliferation and differentiation. Although studies have indicated that soluble mediators such as cytokines and growth factors play a significant role in proliferation and differentiation of stem cells, a critical role for mechanical force and mechanotransduction in controlling self-renewal, differentiation, aging and apoptosis of stem cells has also been revealed. In this review, we summarize and discuss the mechanisms of cells response to mechanical stimuli, the biomechanical force as a regulator for stem cell self-renewal and differentiation, and the putative mechanisms of mechanotransduction.

Bottom-up signaling from HGF-containing surfaces promotes hepatic differentiation of mesenchymal stem cells

The capacity of stem cells to differentiate into specific cell types makes them very promising in tissue regeneration and repair. However, realizing this promise requires novel methods for guiding lineage-specific differentiation of stem cells. In this study, hepatocyte growth factor (HGF), an important morphogen in liver development, was co-printed with collagen I (Col) to create arrays of protein spots on glass. Human adipose stem cells (ASCs) were cultured on top of the HGF/Col spots for 2 weeks. The effects of surface-immobilized HGF on hepatic differentiation of ASCs were analyzed using RT-PCR, ELISA and immunocytochemistry. Stimulation of stem cells with HGF from the bottom-up caused an upregulation in synthesis of α-fetoprotein and albumin, as determined by immunocytochemistry and ELISA. RT-PCR results showed that the mRNA levels for albumin, α-fetoprotein and α1-antitrypsin were 10- to 20-fold higher in stem cells cultured on the HGF/Col arrays compared to stem cells on Col only spots. Our results show that surfaces containing HGF co-printed with ECM proteins may be used to differentiate mesenchymal stem cells such as ASCs into hepatocyte-like cells. These results underscore the utility of growth factor-containing culture surfaces for stem cell differentiation.

Differentiation of Stem Cells: Strategies for Modifying Surface Biomaterials

Stem cells are a natural choice for cellular therapy because of their potential to differentiate into a variety of lineages, their capacity for self-renewal in the repair of damaged organs and tissues in vivo, and their ability to generate tissue constructs in vitro. Determining how to efficiently drive stem cell differentiation to a lineage of choice is critical for the success of cellular therapeutics. Many factors are involved in this process, the extracellular microenvironment playing a significant role in controlling cellular behavior. In recent years, researchers have focused on identifying a variety of biomaterials to provide a microenvironment that is conducive to stem cell growth and differentiation and that ultimately mimics the in vivo situation. Appropriate biomaterials support the cellular attachment, proliferation, and lineage-specific differentiation of stem cells. Tissue engineering approaches have been used to incorporate growth factors and morphogenetic factors-factors known to induce lineage commitment of stem cells-into cultures with scaffolding materials, including synthetic and naturally derived biomaterials. This review focuses on various strategies that have been used in stem cell expansion and examines modifications of natural and synthetic materials, as well as various culture conditions, for the maintenance and lineage-specific differentiation of embryonic and adult stem cells.

Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage

Stem cell response can be influenced by a multitude of chemical, topological and mechanical physiochemical cues. While extensive studies have been focused on the use of soluble factors to direct stem cell differentiation, there are growing evidences illustrating the potential to modulate stem cell differentiation via precise engineering of cell shape. Fibronectin were printed on poly(lactic-co-glycolic acid) (PLGA) thin film forming spatially defined geometries as a means to control the morphology of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs that were cultured on unpatterned substrata adhered and flattened extensively (∼10,000 μm2) while cells grown on 20 μm micropatterend wide adhesive strips were highly elongated with much smaller area coverage of ∼2000 μm2. Gene expression analysis revealed up-regulation of several hallmark markers associated to neurogenesis and myogenesis for cells that were highly elongated while osteogenic markers were specifically down-regulated or remained at its nominal level. Even though there is clearly upregulated levels of both neuronal and myogenic lineages but at the functionally relevant level of protein expression, the myogenic lineage is dominant within the time scale studied as determined by the exclusive expression of cardiac myosin heavy chain for the micropatterned cells. Enforced cell shape distortion resulting in large scale rearrangement of cytoskeletal network and altered nucleus shape has been proposed as a physical impetus by which mechanical deformation is translated into biochemical response. These results demonstrated for the first time that cellular shape modulation in the absence of any induction factors may be a viable strategy to coax lineage-specific differentiation of stem cells.

Biomaterials for stem cell differentiation

Stem cell-based therapeutic treatments hold great promise in treating diseases by replacement of body parts and organs through the generation of new tissues. Directing the controlled differentiation of stem cells into a lineage-specific manner requires a controlled biophysical and biochemical microenvironment and signals. Biomaterials may act as artificial microenvironments and therefore, can deliver stem cell regulatory signals in a precise and near-physiological fashion. A variety of biomaterials are being developed that support cellular attachment, proliferation and most importantly lineage-specific differentiation. Development of new intelligent biomaterials in synergy with cell biology will advance stem cell-based clinical therapeutics. This review discusses the various biomaterials, in particular polymers, applied towards the goal of lineage-specific differentiation of stem cells.

Directing hepatic differentiation of embryonic stem cells with protein microarray-based co-cultures

Embryonic stem cells hold considerable promise in tissue engineering and regenerative medicine as a source of tissue-specific cells. However, realizing this promise requires novel methods for guiding lineage-specific differentiation of stem cells. In this study, we developed a micropatterned co-culture platform for stimulating hepatic differentiation of mouse embryonic stem cells (mESCs). Studies of mESC and hepatic cell adhesion preferences revealed that mESCs required fibronectin for attachment, while hepatic cells (HepG2) preferred collagen (I) substrate and did not adhere to fibronectin. Printing columns of collagen (I) and fibronectin spots (300 microm diameter), followed by sequential seeding of the two cell types, allowed the positioning of clusters of mESCs adjacent to groups of hepatic cells within the same microarray. These micropatterned co-cultures were maintained for up to two weeks in hepatic differentiation media supplemented. To examine the differentiation, mESCs were selectively extracted from the co-culture using laser microdissection and analyzed using real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). These analyses revealed that mESCs co-cultured with HepG2 cells showed a decrease in pluripotency gene expression concomitant with up-regulation of endodermal genes. In addition, the co-culture format induced a significant increase in the expression of liver genes compared to mESCs cultured alone. In conclusion, micropatterned co-cultures of mESCs and hepatic cells showed a significant promise in driving stem cell differentiation towards hepatic phenotype. In the future, this cell culture platform will be further enhanced to enable efficient conversion of mouse and human ESCs to hepatocytes.