Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2016)

The derivation of human induced pluripotent stem cells (IPSCs) has revolutionized the field of personalized medicine. With their ability to self-renew and differentiate to all cell types of the adult body, their therapeutic potential for multiple diseases is invaluable. Albeit their therapeutic potential is promising, they pose the risk of uncontrolled differentiation or tumorigenesis. Physical forces in the developing embryo have been shown to play a critical role in driving lineage commitment to regulate tissue morphogenesis. In this regard, the current work focused on examining the effects of the mechanical microenvironment, using electrospun scaffolds, on the cellular behaviors of human IPSCs. We examined the role of scaffold stiffness on the (1) self-renewal and (2) directed differentiation of IPSCs. Our findings demonstrate that the mechanical microenvironment contributes to the development of distinct IPSC colony morphology. Under proliferation conditions, a two-dimensional (2D) colony morphology on stiff scaffolds enhances proliferation and minimizes spontaneous differentiation of IPSCs. In contrast, the development of three-dimensional (3D) colonies on soft scaffolds results in increased spontaneous differentiation towards ectodermal lineage. Additionally, the development of distinct colonies, directed differentiation to mesendodermal lineage is enhanced on stiff scaffolds while ectodermal differentiation is enhanced on soft scaffolds. Furthermore, we demonstrate that modulation of the RhoA signaling pathway by scaffold stiffness or using a ROCK inhibitor can prime IPSCs to differentiate towards a mesendodermal lineage. We further demonstrate that actin and E-cadherin/β-catenin clustering mediates structural pre-stresses imposed on the cells, which may delay activation of the Wnt signaling pathway, as a result of developing 3D colony morphologies. Finally, we demonstrate that modulation of the scaffold stiffness, at each developmental stage of differentiation towards three germ layer derivate cell phenotypes, can enhance the differentiation of IPSCs. Overall, these findings demonstrate that IPSCs are mechano-responsive and that the mechanical niche can be designed to modulate IPSC behaviors for the control of differentiation or to study developmental processes in vitro.

    Cover page: Regulation of the Mechanical Niche to Guide Pluripotent Stem Cell Behaviors
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2013)
    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.
      Cover page: The Role of Changes in Extracellular Matrix of Cartilage in the Presence of Inflammation on the Pathology of Osteoarthritis
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2016)
      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.
        Cover page: ROCK inhibitor primes human induced pluripotent stem cells to selectively differentiate towards mesendodermal lineage via epithelial-mesenchymal transition-like modulation
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2012)
        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.
          Cover page: Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2018)
          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.
            Cover page: Magnitude‐dependent and inversely‐related osteogenic/chondrogenic differentiation of human mesenchymal stem cells under dynamic compressive strain
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2017)

            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.
              Cover page: Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2017)
              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.
                Cover page: Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2024)
                The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.
                  Cover page: Nanofiber-microwell cell culture system for spatially patterned differentiation of pluripotent stem cells in 3DCreative Commons 'BY-NC-ND' version 4.0 license
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2023)
                  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 β1 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 β1 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.
                    Cover page: Coordination between ECM and cell-cell adhesion regulates the development of islet aggregation, architecture, and functional maturation
                    Top