Direct Stem Cell Differentiation Research Articles

Engineered substrates with imprinted cell-like topographies induce direct differentiation of adipose-derived mesenchymal stem cells into Schwann cells

Differentiation of stem cells to Schwann is considered efficient way for nerve regeneration since the sources of human Schwann cells are limited for clinical application. It is demonstrated that mimicking micromechanical forces or micro/nanotopographical environments that stem cells are experienced in vivo could control their fate. Here, the potency of substrates with imprinted cell-like topographies for direct differentiation of adipose-derived mesenchymal stem cells (ADSCs) into Schwann cells (SCs) is reported. For the preparation of substrates with imprinted SC-Like topographies, SCs are isolated from the sciatic nerve, grown, fixed, and then SC morphologies are transferred to polydimethylsiloxane (PDMS) substrates by mold casting. Subsequently, mesenchymal stem cells (MSCs) are seeded on the SC-imprinted substrates and their differentiation to SCs is evaluated by immunocytochemistry, real-time PCR, and western blotting. Analysis of morphology and expression of SC-specific markers show that MSCs cultured on the imprinted substrates have the typical SC-like morphology and express SC-specific markers including S100b, p75NTR, and Sox10. It is believed that specific cell-like topographies and related micromechanical cues can be sufficient for direct differentiation of ADSCs into Schwann cells by cell-imprinting method as a physical technique.

Combining Stem Cells and Biomaterial Scaffolds for Constructing Tissues and Cell Delivery

Combining stem cells with biomaterial scaffolds serves as a promising strategy for engineering tissues for both in vitro and in vivo applications. This updated review details commonly used biomaterial scaffolds for engineering tissues from stem cells. We first define the different types of stem cells and their relevant properties and commonly used scaffold formulations. Next, we discuss natural and synthetic scaffold materials typically used when engineering tissues, along with their associated advantages and drawbacks and gives examples of target applications. New approaches to engineering tissues, such as 3D bioprinting, are described as they provide exciting opportunities for future work along with current challenges that must be addressed. Thus, this review provides an overview of the available biomaterials for directing stem cell differentiation as a means of producing replacements for diseased or damaged tissues.

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 ZTFn , is presented that closely mimics human plasma fibronectin and serves as an economical, xeno-free, biodegradable, and functionally adaptable cell substrate. The ZTFn substrate supports with high performance the propagation and long-term self-renewal of human embryonic stem cells while preserving their pluripotency. The ZTFn 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.

Development of Hematopoietic Stem Cells in the Early Mammalian Embryo.

Hematopoietic stem cells (HSCs) were the first stem cells discovered in humans. A. A. Maximov proposed an idea of blood stem cells that was confirmed later by McCulloch and Till experimentally. HSCs were the first type of stem cells to be used in clinics and ever since are being continually used. Indeed, a single HSC transplanted intravenously is capable of giving rise to all types of blood cells. In recent decades, human and animal HSC origin, development, hierarchy, and gene signature have been extensively investigated. Due to the constant need for donor blood and HSCs suitable for therapeutic transplants, the experimental possibility of obtaining HSCs in vitro by directed differentiation of pluripotent stem cells (PSCs) has been considered in recent years. However, despite all efforts, it is not yet possible to reproduce in vitro the ontogenesis of HSCs and obtain cells capable of long-term maintenance of hematopoiesis. The study of hematopoiesis in embryonic development facilitates the establishment and improvement of protocols for deriving blood cells from PCSs and allows a better understanding of the pathogenesis of various types of proliferative blood diseases, anemia, and immunodeficiency. This review focuses on the development of hematopoiesis in mammalian ontogenesis.

Utility of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for an In Vitro Model of Proliferative Vitreoretinopathy.

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.

Microhexagon gradient array directs spatial diversification of spinal motor neurons.

Motor neuron diversification and regionalization are important hallmarks of spinal cord development and rely on fine spatiotemporal release of molecular cues. Here, we present a dedicated platform to engineer complex molecular profiles for directed neuronal differentiation.Methods: The technology, termed microhexagon interlace for generation of versatile and fine gradients (microHIVE), leverages on an interlocking honeycomb lattice of microstructures to dynamically pattern molecular profiles at a high spatial resolution. By packing the microhexagons as a divergent, mirrored array, the platform not only enables maximal mixing efficiency but also maintains a small device footprint.Results: Employing the microHIVE platform, we developed optimized profiles of growth factors to induce rostral-caudal patterning of spinal motor neurons, and directed stem cell differentiation in situ into a spatial continuum of different motor neuron subtypes.Conclusions: The differentiated cells showed progressive RNA and protein signatures, consistent with that of representative brachial, thoracic and lumbar regions of the human spinal cord. The microHIVE platform can thus be utilized to develop advanced biomimetic systems for the study of diseases in vitro.

Micro-scaled topographies direct differentiation of human epidermal stem cells

Human epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells. Statement of SignificanceThe epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.

Bacteriophage-based biomaterials for tissue regeneration.

Bacteriophage, also called phage, is a human-safe bacteria-specific virus. It is a monodisperse biological nanostructure made of proteins (forming the outside surface) and nucleic acids (encased in the protein capsid). Among different types of phages, filamentous phages have received great attention in tissue regeneration research due to their unique nanofiber-like morphology. They can be produced in an error-free format, self-assemble into ordered scaffolds, display multiple signaling peptides site-specifically, and serve as a platform for identifying novel signaling or homing peptides. They can direct stem cell differentiation into specific cell types when they are organized into proper patterns or display suitable peptides. These unusual features have allowed scientists to employ them to regenerate a variety of tissues, including bone, nerves, cartilage, skin, and heart. This review will summarize the progress in the field of phage-based tissue regeneration and the future directions in this field.

From Drosophila segmentation to human cancer therapy.

First described in Drosophila, Hedgehog signalling is a key regulator of embryonic development and tissue homeostasis and its dysfunction underlies a variety of human congenital anomalies and diseases. Although now recognised as a major target for cancer therapy as well as a mediator of directed stem cell differentiation, the unveiling of the function and mechanisms of Hedgehog signalling was driven largely by an interest in basic developmental biology rather than clinical need. Here, I describe how curiosity about embryonic patterning led to the identification of the family of Hedgehog signalling proteins and the pathway that transduces their activity, and ultimately to the development of drugs that block this pathway.

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo.

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.

Long noncoding RNA C21orf121/bone morphogenetic protein 2/microRNA-140-5p gene network promotes directed differentiation of stem cells from human exfoliated deciduous teeth to neuronal cells.

Previous studies have revealed that long noncoding RNA (lncRNA) and microRNAplay a crucial role in autism, which is a childhood neurodevelopmental disorder with complicated genetic origins. Hence, the study concerns whether lncRNA C21orf121/bone morphogenetic proteins 2 (BMP2)/miR-140-5p gene network affects directed differentiation of stem cells from human exfoliated deciduous teeth (SHED) to neuronal cells in rats with autism. Autism models were successfully established. The neuron cells that differentiated from SHED cell were identified. The expression of lncRNA C21orf121, miR-140-5p, BMP2, Nestin, βIII-tubulin, and microtubule-associated protein 2 (MAP2) and the expression of neuron-specific enolase (NSE) were examined. Besides, the gap junction (GJ) function of SHED, the intracellular free Ca 2+ concentration, and the social behavior and repetitive stereotyped movements of rats in autism were detected. The target relationship between lncRNA C21orf121 and miR-140-5p and that between miR-140-5p and BMP2 were also verified. Firstly, we successfully isolated SHED and identified the differentiated neurons of SHED. Besides, the expression of BMP2, MAP2, Nestin, βIII-tubulin, NSE positive rate, GJ function, and intracellular free Ca 2+ concentration were increased with the upregulation of C21orf121 and downregulation of miR-140-5p, and accumulated time of repetitive stereotyped movements decreased and the frequency of social behavior increased. The results indicate that lncRNA C21orf121 as a competing endogenous RNAcompetes with BMP2 binding to miR-140-5p, thereby promoting SHED to differentiate into neuronal cells via upregulating BMP2 expression.

Tissue-specific extracellular matrix scaffolds for the regeneration of spatially complex musculoskeletal tissues

Biological scaffolds generated from tissue-derived extracellular matrix (ECM) are commonly used clinically for soft tissue regeneration. Such biomaterials can enhance tissue-specific differentiation of adult stem cells, suggesting that structuring different ECMs into multi-layered scaffolds can form the basis of new strategies for regenerating damaged interfacial tissues such as the osteochondral unit. In this study, mass spectrometry is used to demonstrate that growth plate (GP) and articular cartilage (AC) ECMs contain a unique array of regulatory proteins that may be particularly suited to bone and cartilage repair respectively. Applying a novel iterative freeze-drying method, porous bi-phasic scaffolds composed of GP ECM overlaid by AC ECM are fabricated, which are capable of spatially directing stem cell differentiation in vitro, promoting the development of graded tissues transitioning from calcified cartilage to hyaline-like cartilage. Evaluating repair 12-months post-implantation into critically-sized caprine osteochondral defects reveals that these scaffolds promote regeneration in a manner distinct to commercial control-scaffolds. The GP layer supports endochondral bone formation, while the AC layer stimulates the formation of an overlying layer of hyaline cartilage with a collagen fiber architecture better recapitulating the native tissue. These findings support the use of a bi-layered, tissue-specific ECM derived scaffolds for regenerating spatially complex musculoskeletal tissues.

Saturated fatty acid alters embryonic cortical neurogenesis through modulation of gene expression in neural stem cells

A perturbed maternal metabolic environment such as chronically elevated circulating free fatty acids have been shown to affect stem cell fate during embryonic neurogenesis. However, molecular mechanisms behind this are not well defined, especially in human. Here in using directed differentiation of human embryonic stem cells (hESCs) into cortical neurons as model, we show that chronically elevated saturated fatty acid (palmitate) results in decreased proliferation of neural stem cells and increased differentiation into neurons. This phenotype could be due to palmitate mediated increased expression of key genes needed for neuronal differentiation such as EOMES, TBR1, NEUROD1 and RELN and reduced expression of SREBP regulated lipogenic genes at early stages of cortical differentiation. Furthermore, palmitate treatment increased histone acetylation globally and at select gene promoters among affected genes. We also found differential expression of several lncRNAs associated with cellular stress and metabolic diseases in the presence of palmitate including BDNF-AS suggesting the contribution of additional epigenetic regulatory mechanisms. Together, our results show that saturated fatty acid affects developmental neurogenesis through modulation of gene expression and through epigenetic regulatory mechanisms.

Emerging Strategies for Stem Cell Lineage Commitment in Tissue Engineering and Regenerative Medicine.

Stem cells have transformed the fields of tissue engineering and regenerative medicine, and their potential to further advance these fields cannot be overstated. The stem cell niche is a dynamic microenvironment that determines cell fate during development and tissue repair following an injury. Classically, stem cells were studied in isolation of their microenvironment; however, contemporary research has produced a myriad of evidence that shows the importance of multiple aspects of the stem cell niche in regulating their processes. In the context of tissue engineering and regenerative medicine studies, the niche is an artificial environment provided by culture conditions. In vitro culture conditions may involve coculturing with other cell types, developing specific biomaterials, and applying relevant forces to promote the desired lineage commitment. Considerable advance has been made over the past few years toward directed stem cell differentiation; however, the unspecific differentiation of stem cells yielding a mixed population of cells has been a challenge. In this review, we provide a systematic review of the emerging strategies used for lineage commitment within the context of tissue engineering and regenerative medicine. These strategies include scaffold pore-size and pore-shape gradients, stress relaxation, sonic and electromagnetic effects, and magnetic forces. Finally, we provide insights and perspectives into future directions focusing on signaling pathways activated during lineage commitment using external stimuli.

Nanofibers as Bioinstructive Scaffolds Capable of Modulating Differentiation through Mechanosensitive Pathways for Regenerative Engineering.

Bioinstructive scaffolds encode information in the physical shape and size of materials to direct cell responses. Electrospinning nanofibers is a process that offers control over scaffold architecture and fiber diameter, while providing extended linear length of fibers. This review summarizes tissue engineering literature that has utilized nanofiber scaffolds to direct stem cell differentiation for various tissues including musculoskeletal, vascular, immunological and nervous system tissues. Nanofibers are also considered for their extracellular matrix mimetic characteristics that can preserve stem cell differentiation capacity. These topics are considered in the context of focal adhesion and integrin signaling. Regenerative engineering will be enhanced by construction of scaffolds encoded with shape information to cause an attached cell to create the intended tissue at that region. Nanofibers are likely to be a bioinstructive scaffold in future regenerative engineering development as we pursue the Grand Challenges of engineering tissues.

Transplantation of human intestinal organoids into the mouse mesentery: A more physiologic and anatomic engraftment site

BackgroundWe previously described the development of human intestinal organoids from pluripotent stem cells, as well as their in vivo maturation when transplanted into the mouse kidney capsule. While sufficient for certain aspects of study, this model has limitations. Herein, we describe an alternative model of human intestinal organoids transplantation into the mouse mesentery. We hypothesize that efficient engraftment and marked differentiation of human intestinal organoids will be similar to our kidney model yet in a more anatomically appropriate location allowing for improved in vivo modeling. MethodsHuman intestinal organoids were generated by directed differentiation of H1 embryonic stem cells. Human intestinal organoids were then transplanted into the mesentery of immunosuppressed mice. Gross and histologic analysis of tissue was performed. ResultsHuman intestinal organoids were transplanted into the mouse mesentery and allowed to grow for 10 weeks. Mouse survival was 85%, and among the surviving mice, 82% of transplanted human intestinal organoids successfully engrafted. Upon graft harvest, transplanted HIOs were larger than in vitro human intestinal organoids (1.75 mm vs 6.27 mm, P < .0001) and grew along a vascular pedicle, allowing for interventions and reconstructive surgeries to access the human intestinal organoid lumen. Histologic analyses of transplanted human intestinal organoids confirmed the presence of major cell types, as well as stem cell activity. ConclusionsThe mouse mesentery is a viable location for the transplantation of human intestinal organoids, yielding grafts of reproducible size and quality. This improved model serves to advance functional and translational studies of human intestinal organoids.

A single cell transcriptional portrait of embryoid body differentiation and comparison to progenitors of the developing embryo

Directed differentiation of pluripotent stem cells provides an accessible system to model development. However, the distinct cell types that emerge, their dynamics, and their relationship to progenitors in the early embryo has been difficult to decipher because of the cellular heterogeneity inherent to differentiation. Here, we used a combination of bulk RNA-Seq, single cell RNA-Seq, and bioinformatics analyses to dissect the cell types that emerge during directed differentiation of mouse embryonic stem cells as embryoid bodies and we compared them to spatially and temporally resolved transcriptional profiles of early embryos. Our single cell analyses of the day 4 embryoid bodies revealed three populations which had retained related yet distinct pluripotent signatures that resemble the pre- or post-implantation epiblast, one population of presumptive neuroectoderm, one population of mesendoderm, and four populations of neural progenitors. By day 6, the neural progenitors predominated the embryoid bodies, but both a small population of pluripotent-like cells and an anterior mesoderm-like Brachyury-expressing population were present. By comparing the day 4 and day 6 populations, we identified candidate differentiation paths, transcription factors, and signaling pathways that mark the in vitro correlate of the transition from the mid-to-late primitive streak stage.

Vascular extracellular matrix and fibroblasts-coculture directed differentiation of human mesenchymal stem cells toward smooth muscle-like cells for vascular tissue engineering

Construction of an artificial vascular graft is widely considered a promising strategy in vascular tissue engineering. However, limited sources of functional vascular smooth muscle cells (VSMCs) remain a major obstacle in vascular tissue engineering. In this study, we innovatively developed an approach to obtain functional VSMCs by onsite differentiating human bone marrow-derived mesenchymal stem cells (MSCs) directed by decellularized extracellular matrix (ECM) and fibroblasts. The resulting cells and ECM-cells constructs were characterized by real time RT-PCR, immunofluorescence staining, cell contractile functions, and migration capacity. Our results showed both ECM and fibroblasts induced MSCs differentiation toward VSMC-like cells with increased transcription of marker genes, upregulated expression of contractile apparatus proteins, and enhanced functional activity of VSMC phenotype. Interestingly, our findings revealed that native ECM and fibroblasts-coculture had a higher potential to promote MSCs differentiation into VSMCs than growth factors cocktail (GFC) supplemented culture, thereby providing a potential source of VSMCs for blood vessel constitution.

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.

The Efficacy of Graphene Foams for Culturing Mesenchymal Stem Cells and Their Differentiation into Dopaminergic Neurons.

The implantation of stem cells in vivo is the ideal approach for the restoration of normal life functions, such as replenishing the decreasing levels of affected dopaminergic (DA) neurons during neurodegenerative disease conditions. However, combining stem cells with biomaterial scaffolds provides a promising strategy for engineering tissues or cellular delivery for directed stem cell differentiation as a means of replacing diseased/damaged tissues. In this study, mouse mesenchymal stem cells (MSCs) were differentiated into DA neurons using sonic hedgehog, fibroblast growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor, while they were cultured within collagen-coated 3D graphene foams (GF). The differentiation into DA neurons within the collagen-coated GF and controls (collagen gels, plastic) was confirmed using β-III tubulin, tyrosine hydroxylase (TH), and NeuN positive immunostaining. Enhanced expression of β-III tubulin, TH, and NeuN and an increase in the average neurite extension length were observed when cells were differentiated within collagen-coated GF in comparison with collagen gels. Furthermore, these graphene-based scaffolds were not cytotoxic as MSC seemed to retain viability and proliferated substantially during in vitro culture. In summary, these results suggest the utility of 3D graphene foams towards the differentiation of DA neurons from MSC, which is an important step for neural tissue engineering applications.