Coordination between ECM and cell-cell adhesion regulates the development of islet aggregation, architecture, and functional maturation.

Pancreatic islets are three-dimensional cell aggregates consisting of unique cellular composition, cell-to-cell contacts, and interactions with blood vessels. Cell aggregation is essential for islet endocrine function; however, it remains unclear how developing islets establish aggregation. By combining genetic animal models, imaging tools, and gene expression profiling, we demonstrate that islet aggregation is regulated by extracellular matrix signaling and cell-cell adhesion. Islet endocrine cell-specific inactivation of extracellular matrix receptor integrin ß1 disrupted blood vessel interactions but promoted cell-cell adhesion and the formation of larger islets. In contrast, ablation of cell-cell adhesion molecule α-catenin promoted blood vessel interactions yet compromised islet clustering. Simultaneous removal of integrin ß1 and α-catenin disrupts islet aggregation and the endocrine cell maturation process, demonstrating that establishment of islet aggregates is essential for functional maturation. Our study provides new insights into understanding the fundamental self-organizing mechanism for islet aggregation, architecture, and functional maturation.

Painting the Pancreas in Three Dimensions: Whole-Mount Immunofluorescence Method.

The pancreas is composed of different cellular populations, organized into distinct functional units, including acinar clusters, islets of Langerhans, and the ductal system. As a result of research into diabetes, several optical techniques have been developed for the three-dimensional visualization of islet populations, so as to better understand their anatomical characteristics. These approaches are largely reliant on three-dimensional whole-mount immunofluorescence staining. In this chapter, we review a revised whole mount immunofluorescence staining method for studying adult pancreatic islet morphology. This method uses smaller samples and combines the blocking and permeabilization steps. This reduces the time needed, relative to existing protocols; the method is compatible with regular confocal microscopy as well.

Spatially Regulated Multiphenotypic Differentiation of Stem Cells in 3D via Engineered Mechanical Gradient.

Within the osteochondral interface, cellular and extracellular matrix gradients provide a biomechanical and biochemical niche for homeostatic tissue functions. Postnatal joint loading is critical for the development of such tissue gradients, leading to the formation of functional osteochondral tissues composed of superficial, middle, and deep zones of cartilage, and underlying subchondral bone, in a depth-dependent manner. In this regard, a novel, variable core-shell electrospinning strategy was employed to generate spatially controlled strain gradients within three-dimensional scaffolds under dynamic compressive loading, enabling the local strain-magnitude dependent, multiphenotypic stem cell differentiation. Human mesenchymal stem cells (hMSCs) were cultured in electrospun scaffolds with a linear or biphasic mechanical gradient, which was computationally engineered and experimentally validated. The cell/scaffold constructs were subjected to various magnitudes of dynamic compressive strains in a scaffold depth-dependent manner at a frequency of 1 Hz for 2 h daily for up to 42 days in osteogenic media. Spatially upregulated gene expression of chondrogenic markers (ACAN, COL2A1, PRG4) and glycosaminoglycan deposition was observed in the areas of greater compressive strains. In contrast, osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition were downregulated in response to high local compressive strains. Dynamic mechanical analysis showed the maintenance of the engineered mechanical gradients only under dynamic culture conditions, confirming the potent role of biomechanical gradients in developing and maintaining a tissue gradient. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by regulating local mechanical microenvironments, providing a novel strategy to recapitulate the gradient structure in osteochondral tissues for successful regeneration of damaged joints in vivo and facile development of interfacial tissue models in vitro.

Magnitude-dependent and inversely-related osteogenic/chondrogenic differentiation of human mesenchymal stem cells under dynamic compressive strain.

Biomechanical forces have been shown to significantly affect tissue development, morphogenesis, pathogenesis and healing, especially in orthopaedic tissues. Such biological processes are critically related to the differentiation of human mesenchymal stem cells (hMSCs). However, the mechanistic details regarding how mechanical forces direct MSC differentiation and subsequent tissue formation are still elusive. Electrospun three-dimensional scaffolds were used to culture and subject hMSCs to various magnitudes of dynamic compressive strains at 5, 10, 15 or 20% (ε = 0.05, 0.10, 0.15, 0.20) at a frequency of 1 Hz for 2 h daily for up to 28 days in osteogenic media. Gene expression of chondrogenic markers (ACAN, COL2A1, SOX9) and glycosaminoglycan (GAG) synthesis were upregulated in response to the increased magnitudes of compressive strain, whereas osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition had noticeable decreases by compressive loading in a magnitude-dependent manner. Dynamic mechanical analysis showed enhanced viscoelastic modulus with respect to the increased dynamic strain peaking at 15%, which coincides with the maximal GAG synthesis. Furthermore, polarization-sensitive optical coherence tomography revealed that mechanical loading enhanced the alignment of extracellular matrix to the greatest level by 15% strain as well. Overall, we show that the degree of differentiation of hMSCs towards osteogenic or chondrogenic lineage is inversely related, and it depends on the magnitude of dynamic compressive strain. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by varying the strain regimens, providing a novel strategy to modulate differentiation specification and tissue morphogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation.

BACKGROUND: To maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate stiffness affects the step-wise differentiation of iPSCs towards various lineages/phenotypes. METHODS: Electrospun nanofibrous substrates with different reduced Young's modulus were utilized to subject cells to different mechanical environments during the differentiation process towards representative phenotypes from each of three germ layer derivatives including motor neuron, pancreatic endoderm, and chondrocyte. Phenotype-specific markers of each lineage/stage were utilized to determine differentiation efficiency by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging for gene and protein expression analysis, respectively. RESULTS: The results presented in this proof-of-concept study are the first to systematically demonstrate the significant role of the temporally varied mechanical microenvironment on the differentiation of stem cells. Our results demonstrate that the process of differentiation from pluripotent cells to functional end-phenotypes is mechanoresponsive in a lineage- and differentiation stage-specific manner. CONCLUSIONS: Lineage/developmental stage-dependent optimization of electrospun substrate stiffness provides a unique opportunity to enhance differentiation efficiency of iPSCs for their facilitated therapeutic applications.

Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System.

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study, a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time, in relation to cellular activities during self-renewal or lineage-specific differentiation, in a non-destructive, label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal, or subjected to mesendodermal or ectodermal differentiation, and correlating them to morphological (size, shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall, the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation.

ROCK inhibitor primes human induced pluripotent stem cells to selectively differentiate towards mesendodermal lineage via epithelial-mesenchymal transition-like modulation.

Robust control of human induced pluripotent stem cell (hIPSC) differentiation is essential to realize its patient-tailored therapeutic potential. Here, we demonstrate a novel application of Y-27632, a small molecule Rho-associated protein kinase (ROCK) inhibitor, to significantly influence the differentiation of hIPSCs in a lineage-specific manner. The application of Y-27632 to hIPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration and time-dependent manner. Such changes in cell and colony morphology were associated with decreased expression of E-cadherin, a cell-cell junctional protein, proportional to the increased exposure to Y-27632. Interestingly, gene and protein expression of pluripotency markers such as NANOG and OCT4 were not downregulated by an exposure to Y-27632 up to 36h. Simultaneously, epithelial-to-mesenchymal (EMT) transition markers were upregulated with an exposure to Y-27632. These EMT-like changes in the cells with longer exposure to Y-27632 resulted in a significant increase in the subsequent differentiation efficiency towards mesendodermal lineage. In contrast, an inhibitory effect was observed when cells were subjected to ectodermal differentiation after prolonged exposure to Y-27632. Collectively, these results present a novel method for priming hIPSCs to modulate their differentiation potential with a simple application of Y-27632.

Enhanced Lineage-Specific Differentiation Efficiency of Human Induced Pluripotent Stem Cells by Engineering Colony Dimensionality Using Electrospun Scaffolds.

Electrospun scaffolds with varied stiffness promote distinct colony morphology of human induced pluripotent stem cells, which affects their subsequent differentiation. On soft scaffolds, induced pluripotent stem cells develop 3D colonies due to the pliability of the electrospun fibrous networks, leading to greater differentiation tendency to ectodermal lineage.

The effects of electrospun substrate-mediated cell colony morphology on the self-renewal of human induced pluripotent stem cells.

The development of xeno-free, chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard, various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal, i.e., proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly, when surface chemistry of the substrates was uniformly controlled by collagen conjugation, the stiffness of substrate was inversely related to the sphericity, a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture, implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall, we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal.

The role of changes in extracellular matrix of cartilage in the presence of inflammation on the pathology of osteoarthritis.

Osteoarthritis (OA) is a degenerative disease that affects various tissues surrounding joints such as articular cartilage, subchondral bone, synovial membrane, and ligaments. No therapy is currently available to completely prevent the initiation or progression of the disease partly due to poor understanding of the mechanisms of the disease pathology. Cartilage is the main tissue afflicted by OA, and chondrocytes, the sole cellular component in the tissue, actively participate in the degeneration process. Multiple factors affect the development and progression of OA including inflammation that is sustained during the progression of the disease and alteration in biomechanical conditions due to wear and tear or trauma in cartilage. During the progression of OA, extracellular matrix (ECM) of cartilage is actively remodeled by chondrocytes under inflammatory conditions. This alteration of ECM, in turn, changes the biomechanical environment of chondrocytes, which further drives the progression of the disease in the presence of inflammation. The changes in ECM composition and structure also prevent participation of mesenchymal stem cells in the repair process by inhibiting their chondrogenic differentiation. This review focuses on how inflammation-induced ECM remodeling disturbs cellular activities to prevent self-regeneration of cartilage in the pathology of OA.

Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases.

Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.

Differential uptake of DNA-poly(ethylenimine) polyplexes in cells cultured on collagen and fibronectin surfaces.

Genetically modified bone marrow-derived mesenchymal stem cells (MSCs) have proven to be efficient cell carriers for local or systemic delivery of therapeutics as well as growth factors to augment tissue formation. However, efficient non-viral gene transfer to these cells is limiting their applicability. Although most studies have focused on designing more efficient condensation agents for DNA, our focus in this manuscript is to study the role of two extracellular matrix (ECM) proteins, collagen I (Col I) and fibronectin (Fn), on the ability of MSCs to become transfected. Here we report that plating MSCs on Col I-coated surfaces inhibits transfection, while plating MSCs on Fn-coated surfaces enhances transfection. The mechanism by which these ECM proteins affect non-viral gene transfer involves the endocytosis pathway used for polyplex uptake and intracellular tension. We found that Fn promoted internalization through clathrin-mediated endocytosis and that this pathway resulted in more efficient transfection than caveolae-mediated endocytosis and macropinocytosis. Further, the disruption of actin-myosin interactions resulted in an enhancement of gene transfer for cells plated on Fn-coated surfaces, but not for cells plated on Col I. We believe that the cellular microenvironment can be engineered to enhance the ability of cells to become transfected and that through understanding the mechanisms by which the ECM affects non-viral gene transfer better materials and transfection protocols can be realized.