Characterization of cell signaling,morphology, and differentiation potential of human mesenchymal stem cells based on cell adhesion mechanism

It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose-binding protein (MBP)-fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin-independent heparin sulfate proteoglycans-mediated cell adhesion. In this study, we characterize cell adhesion-dependent cellular behaviors of human adipose-derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP-fused basic fibroblast growth factor (MF)-coated surface and fibronectin (FN)-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.     

A role for chemistry in stem cell biology

Although stem cells hold considerable promise for the treatment of numerous diseases including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes and cancer, obstacles such as the control of stem cell fate, allogenic rejection and limited cell availability must be overcome before their therapeutic potential can be realized. This requires an improved understanding of the signaling pathways that affect stem cell fate. Cell-based phenotypic and pathway-specific screens of natural products and synthetic compounds have recently provided a number of small molecules that can be used to selectively control stem cell proliferation and differentiation. Examples include the selective induction of neurogenesis and cardiomyogenesis in murine embryonic stem cells, osteogenesis in mesenchymal stem cells and dedifferentiation in skeletal muscle cells. Such molecules will likely provide new insights into stem cell biology, and may ultimately contribute to effective medicines for tissue repair and regeneration.     

New materials for tissue engineering: towards greater control over the biological response

One goal of tissue engineering is to replace lost or compromised tissue function, and an approach to this is to control the interplay between materials (scaffolds), cells and growth factors to create environments that promote the regeneration of functional tissues and organs. An increased understanding of the chemical signals that direct cell differentiation, migration and proliferation, advances in scaffold design and peptide engineering that allow this signaling to be recapitulated and the development of new materials, such as DNA-based and stimuli-sensitive polymers, have recently given engineers enhanced control over the chemical properties of a material and cell fate. Additionally, the immune system, which is often overlooked, has been shown to play a beneficial role in tissue repair, and future endeavors in material design will potentially expand to include immunomodulation.     

Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.     

Harnessing pluripotency from differentiated cells: a regenerative source for tissue-specific stem cell therapies

Processes involving conversion of mature adult cells into undifferentiated cells have tremendous therapeutic potential in treating a variety of malignant and non-malignant disorders, including degenerative diseases. This can be achieved in autologous or allogeneic settings, by replacing either defective cells or regenerating those that are in deficit through reprogramming more committed cells into stem cells. The concept behind reprogramming differentiated cells to a stem cell state is to enable the switching of development towards the required cell lineage that is capable of correcting the underlying cellular dysfunction. The techniques by which differentiated cells can reverse their development, become pluripotent stem cells and transdifferentiate to give rise to new tissue or an entire organism are currently under intense investigation. Examples of reprogramming differentiation in mature adult cells include nuclear reprogramming of more committed cells using the cytoplasm of empty oocytes obtained from a variety of animal species, or cell surface contact of differentiated cells through receptor ligand interaction. Such ligands include monoclonal antibodies, cytokines or synthetic chemical compounds. Despite controversies surrounding such techniques, the concept behind identification and design/screening of biological or pharmacological compounds to enable re-switching of cell fate in-vivo or ex-vivo is paramount for current drug therapies to be able to target more specifically cellular dysfunction at the tissue/organ level. Herein, this review discusses current research in cellular reprogramming and its potential application in regenerative medicine.     

Geometric control of myogenic cell fate

This work combines expertise in stem cell biology and bioengineering to define the system for geometric control of proliferation and differentiation of myogenic progenitor cells. We have created an artificial niche of myogenic progenitor cells, namely, modified extracellular matrix (ECM) substrates with spatially embedded growth or differentiation factors (GF, DF) that predictably direct muscle cell fate in a geometric pattern. Embedded GF and DF signal progenitor cells from specifically defined areas on the ECM successfully competed against culture media for myogenic cell fate determination at a clearly defined boundary. Differentiation of myoblasts into myotubes is induced in growth-promoting medium, myotube formation is delayed in differentiation-promoting medium, and myogenic cells, at different stages of proliferation and differentiation, can be induced to coexist adjacently in identical culture media. This method can be used to identify molecular interactions between cells in different stages of myogenic differentiation, which are likely to be important determinants of tissue repair. The designed ECM niches can be further developed into a vehicle for transplantation of myogenic progenitor cells maintaining their regenerative potential. Additionally, this work may also serve as a general model to engineer synthetic cellular niches to harness the regenerative potential of organ stem cells.     

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.     

Cell delivery mechanisms for tissue repair

Many cell populations, derived from both adult tissues and embryonic stem cells, show promise for the treatment of a variety of diseases. Although the major effort in stem cell therapies in the past has been identifying potentially therapeutic cells, it is now clear that developing systems to deliver these cells and promote their efficient engraftment will provide an equally challenging task. More sophisticated pretransplantation manipulations and material carriers may dramatically improve the survival, engraftment, and fate control of transplanted stem cells and their ultimate clinical utility.     

Tissue scaffold surface patterning for clinical applications

Patterned scaffold surfaces provide a platform for highly defined cellular interactions, and have recently taken precedence in tissue engineering. Despite advances in patterning techniques and improved tissue growth, no clinical studies have been conducted for implantation of patterned biomaterials. Four major clinical application fields where patterned materials hold great promise are antimicrobial surfaces, cardiac constructs, neurite outgrowth, and stem cell differentiation. Specific examples include applications of patterned materials to (i) counter infection by antibiotic resistant bacteria, (ii) establish proper alignment and contractile force of regrown cardiac cells for repairing tissue damaged by cardiac infarction, (iii) increase neurite outgrowth for central nervous system wound repair, and (iv) host differentiated stem cells while preventing reversion to a pluripotent state. Moreover, patterned materials offer unique advantages for artificial implants which other constructs cannot. For example, by inducing selective cell adhesion using topographical cues, patterned surfaces present cellular orientation signals that lead to functional tissue architectures. Mechanical stimuli such as modulus, tension, and material roughness are known to influence tissue growth, as are chemical stimuli for cell adhesion. Scaffold surface patterns allow for control of these mechanical and chemical factors. This review identifies research advances in scaffold surface patterning, in light of pressing clinical needs requiring organization of cellular interactions.     

The development of high-throughput screening approaches for stem cell engineering

It has become increasingly clear that both soluble factors, such as growth factors, and insoluble factors, including the surfaces on which cells grow, can have controlling effects on stem cell behavior and differentiation. While much progress has been made in biomaterial design and application, the rational design of biomaterial cues to direct stem cell behavior and differentiation remains challenging. Recent advances in automated, high-throughput methods for synthesizing and screening combinatorial biomaterial libraries and cellular microenvironments promise to accelerate the discovery of factors that control stem cell behavior. Specific examples include miniaturized, automated, combinatorial material synthesis and extracellular matrix screening methods as well microarrayed methods for creating local microenvironments of soluble factors, such as small molecules, siRNA, and other signaling molecules.     

Mitochondria and the dynamic control of stem cell homeostasis

The maintenance of cellular identity requires continuous adaptation to environmental changes. This process is particularly critical for stem cells, which need to preserve their differentiation potential over time. Among the mechanisms responsible for regulating cellular homeostatic responses, mitochondria are emerging as key players. Given their dynamic and multifaceted role in energy metabolism, redox, and calcium balance, as well as cell death, mitochondria appear at the interface between environmental cues and the control of epigenetic identity. In this review, we describe how mitochondria have been implicated in the processes of acquisition and loss of stemness, with a specific focus on pluripotency. Dissecting the biological functions of mitochondria in stem cell homeostasis and differentiation will provide essential knowledge to understand the dynamics of cell fate modulation, and to establish improved stem cell‐based medical applications.     

Multifunctionality of PIWI proteins in control of germline stem cell fate

PIWI proteins interacting with specific type of small RNAs (piRNAs) repress transposable elements in animals. Besides, they have been shown to participate in various cellular processes: in the regulation of heterochromatin formation including telomere structures, in the control of translation and the cell cycle, and in DNA rearrangements. PIWI proteins were first identified by their roles in the self-renewal of germline stem cells. PIWI protein functions are not limited to gonadogenesis, but the role in determining the fate of stem cells is their specific feature conserved throughout the evolution of animals. Molecular mechanisms underlying these processes are far from being understood. This review focuses on the role of PIWI proteins in the control of maintenance and proliferation of germinal stem cells and its relation to the known function of PIWI in transposon repression.     

Therapeutic strategies targeting somatic stem cells: Chemical approaches

Therapeutic modulation of fate and behavior of somatic stem cells can generate safe and functional cells ex vivo for cell-based therapy, or to repair and regenerate damaged tissues in vivo. Chemical approaches involving small molecules have provided promising approaches for modulating cellular fate and function. These strategies offer opportunities that support regenerative medicine. Here, we discuss strategies targeting somatic stem cells through chemical approaches, highlighting their progression as well as future prospects.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

Human adult pluripotency: Facts and questions

Cellular reprogramming and induced pluripotent stem cell(IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine.While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium-and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues(such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.     

Direct cell contact influences bone marrow mesenchymal stem cell fate

Adult bone marrow-derived mesenchymal stem cells (MSC) can differentiate into various cell types of mesenchymal origin, but mechanisms regulating such cellular changes are unclear. We have conducted co-culture experiments to examine whether mesenchymal stem cell differentiation is influenced by indirect or direct contact with differentiated cells. Cultured adult mesenchymal stem cells showed some characteristics of synthetic state vascular smooth muscle cells (SMC). When co-cultured with vascular endothelial cells (EC) without cell contact, they exhibited abundant well-organised smooth muscle alpha-actin (alpha-actin) filaments. Direct co-culture with endothelial cells resulted in increased smooth muscle alpha-actin mRNA and protein, yet also comprehensive disruption of smooth muscle alpha-actin filament organisation. In order to assess whether these cell contact effects on mesenchymal stem cells were cell type specific, we also analysed direct co-cultures of mesenchymal stem cells with dermal fibroblasts. However, these experiments were characterised by the appearance of abundant spindle-shaped myofibroblast-like cells containing organised smooth muscle alpha-actin filaments. Thus, direct contact with distinct differentiated cells may be a critical determinant of mesenchymal stem cell fate in blood vessels and other connective tissues.     

Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages

Human pluripotent cells such as human embryonic stem cells (hESC) are a great potential source of cells for cell-based therapies; however, directing their differentiation into the desired cell types with high purity remains a challenge. The stem cell microenvironment plays a vital role in directing hESC fate and we have previously shown that manipulation of colony size in a serum- and cytokine-free environment controls self-renewal and differentiation toward the extraembryonic endoderm lineage. Here we show that, in the presence of bone morphogenetic protein 2 and activin A, control of colony size using a microcontact printing technology is able to direct hESC fate to either the mesoderm or the endoderm lineage. Large, 1200-μm-diameter colonies give rise to mesoderm, while small 200-μm colonies give rise to definitive endoderm. This study links, for the first time, cellular organization to pluripotent cell differentiation along the mesoderm and endoderm lineages.     

Stem cell biology and neurodegenerative disease

The fundamental basis of our work is that organs are generated by multipotent stem cells, whose properties we must understand to control tissue assembly or repair. Central nervous system (CNS) stem cells are now recognized as a well-defined population of precursors that differentiate into cells that are indisputably neurons and glial cells. Work from our group played an important role in defining stem cells of the CNS. Embryonic stem (ES) cells also differentiate to specific neuron and glial types through defined intermediates that are similar to the cellular precursors that normally occur in brain development. There is convincing evidence that the differentiated progeny of ES cells and CNS stem cells show expected functions of neurons and glia. Recent progress has been made on three fundamental developmental processes: (i) cell cycle control; (ii) the control of cell fate; and (iii) early steps in neural differentiation. In addition, our work on CNS stem cells has developed to a stage where there are clinical implications for Parkinson's and other degenerative disorders. These advances establish that stem cell biology contributes to our understanding of brain development and has great clinical promise.