Optical microscopic imaging offers opportunities to perform noninvasive assessments of numerous parameters associated with the biochemistry, morphology, and functional state of biological samples. ...For example, it is possible to detect the endogenous fluorescence from a small number of important biomolecules, including NADH and FAD, which are two coenzymes involved in key metabolic pathways such as glycolysis, the Krebs cycle, and oxidative phosphorylation. Here, we review different imaging approaches to isolate the fluorescence from these chromophores in two- and three-dimensional samples and discuss the origins and potential interpretation of the observed signals in terms of cell metabolic status. Finally, we discuss the challenges and limitations of these approaches, as well as important research directions that we expect will evolve in the near future.
A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we ...report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.
Postnatal bone growth involves a dramatic increase in length and girth. Intriguingly, this period of growth is independent of growth hormone and the underlying mechanism is poorly understood. ...Recently, an
mutation was identified in humans with early postnatal growth restriction. Here, we show that IGF2 is essential for longitudinal and appositional murine postnatal bone development, which involves proper timing of chondrocyte maturation and perichondrial cell differentiation and survival. Importantly, the
null mouse model does not represent a simple delay of growth but instead uncoordinated growth plate development. Furthermore, biochemical and two-photon imaging analyses identified elevated and imbalanced glucose metabolism in the
null mouse. Attenuation of glycolysis rescued the mutant phenotype of premature cartilage maturation, thereby indicating that IGF2 controls bone growth by regulating glucose metabolism in chondrocytes. This work links glucose metabolism with cartilage development and provides insight into the fundamental understanding of human growth abnormalities.
Tendons have uniquely high tensile strength, critical to their function to transfer force from muscle to bone. When injured, their innate healing response results in aberrant matrix organization and ...functional properties. Efforts to regenerate tendon are challenged by limited understanding of its normal development. Consequently, there are few known markers to assess tendon formation and parameters to design tissue engineering scaffolds. We profiled mechanical and biological properties of embryonic tendon and demonstrated functional properties of developing tendon are not wholly reflected by protein expression and tissue morphology. Using force volume-atomic force microscopy, we found that nano- and microscale tendon elastic moduli increase nonlinearly and become increasingly spatially heterogeneous during embryonic development. When we analyzed potential biochemical contributors to modulus, we found statistically significant but weak correlation between elastic modulus and collagen content, and no correlation with DNA or glycosaminoglycan content, indicating there are additional contributors to mechanical properties. To investigate collagen cross-linking as a potential contributor, we inhibited Iysyl oxidase-mediated collagen cross-linking, which significantly reduced tendon elastic modulus without affecting collagen morphology or DNA, glycosaminoglycan, and collagen content. This suggests that Iysyl oxidase-mediated cross-linking plays a significant role in the development of embryonic tendon functional properties and demonstrates that changes in cross-links alter mechanical properties without affecting matrix content and organization. Taken together, these data demonstrate the importance of functional markers to assess tendon development and provide a profile of tenogenic mechanical properties that may be implemented in tissue engineering scaffold design to mechanoregulate new tendon regeneration.
Dynamic alterations in the unique brain extracellular matrix (ECM) are involved in malignant brain tumors. Yet studies of brain ECM roles in tumor cell behavior have been difficult due to lack of ...access to the human brain. We present a tunable 3D bioengineered brain tissue platform by integrating microenvironmental cues of native brain-derived ECMs and live imaging to systematically evaluate patient-derived brain tumor responses. Using pediatric ependymoma and adult glioblastoma as examples, the 3D brain ECM-containing microenvironment with a balance of cell-cell and cell-matrix interactions supports distinctive phenotypes associated with tumor type-specific and ECM-dependent patterns in the tumor cells' transcriptomic and release profiles. Label-free metabolic imaging of the composite model structure identifies metabolically distinct sub-populations within a tumor type and captures extracellular lipid-containing droplets with potential implications in drug response. The versatile bioengineered 3D tumor tissue system sets the stage for mechanistic studies deciphering microenvironmental role in brain tumor progression.
Abstract
Circulating tumor cell clusters (CTCCs) are rare cellular events found in the blood stream of metastatic tumor patients. Despite their scarcity, they represent an increased risk for ...metastasis. Label-free detection methods of these events remain primarily limited to in vitro microfluidic platforms. Here, we expand on the use of confocal backscatter and fluorescence flow cytometry (BSFC) for label-free detection of CTCCs in whole blood using machine learning for peak detection/classification. BSFC uses a custom-built flow cytometer with three excitation wavelengths (405 nm, 488 nm, and 633 nm) and five detectors to detect CTCCs in whole blood based on corresponding scattering and fluorescence signals. In this study, detection of CTCC-associated GFP fluorescence is used as the ground truth to assess the accuracy of endogenous back-scattered light-based CTCC detection in whole blood. Using a machine learning model for peak detection/classification, we demonstrated that the combined use of backscattered signals at the three wavelengths enable detection of ~ 93% of all CTCCs larger than two cells with a purity of > 82% and an overall accuracy of > 95%. The high level of performance established through BSFC and machine learning demonstrates the potential for label-free detection and monitoring of CTCCs in whole blood. Further developments of label-free BSFC to enhance throughput could lead to important applications in the isolation of CTCCs in whole blood with minimal disruption and ultimately their detection in vivo.
The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue ...development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.
Silk fibroin has been successfully used as a biomaterial for tissue regeneration. To prepare silk fibroin biomaterials for human implantation a series of processing steps are required to purify the ...protein. Degumming to remove inflammatory sericin is a crucial step related to biocompatibility and variability in the material. Detailed characterization of silk fibroin degumming is reported. The degumming conditions significantly affected cell viability on the silk fibroin material and the ability to form three-dimensional porous scaffolds from the silk fibroin, but did not affect macrophage activation or β-sheet content in the materials formed. Methods are also provided to determine the content of residual sericin in silk fibroin solutions and to assess changes in silk fibroin molecular weight. Amino acid composition analysis was used to detect sericin residuals in silk solutions with a detection limit between 1.0 and 10% wt/wt, while fluorescence spectroscopy was used to reproducibly distinguish between silk samples with different molecular weights. Both methods are simple and require minimal sample volume, providing useful quality control tools for silk fibroin preparation processes.
The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the ...efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed.
Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress.
In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner.
Cancer cell-secreted eHsp90 binds and activates proteins in the tumor microenvironment crucial in cancer invasion. Therefore, targeting eHsp90 could inhibit invasion, preventing metastasis-the ...leading cause of cancer-related mortality. Previous eHsp90 studies have solely focused on its role in cancer invasion through the 2D basement membrane (BM), a form of extracellular matrix (ECM) that lines the epithelial compartment. However, its role in cancer invasion through the 3D Interstitial Matrix (IM), an ECM beyond the BM, remains unexplored. Using a Collagen-1 binding assay and second harmonic generation (SHG) imaging, we demonstrate that eHsp90 directly binds and aligns Collagen-1 fibers, the primary component of IM. Furthermore, we show that eHsp90 enhances Collagen-1 invasion of breast cancer cells in the Transwell assay. Using Hsp90 conformation mutants and inhibitors, we established that the Hsp90 dimer binds to Collagen-1 via its N-domain. We also demonstrated that while Collagen-1 binding and alignment are not influenced by Hsp90's ATPase activity attributed to the N-domain, its open conformation is crucial for increasing Collagen-1 alignment and promoting breast cancer cell invasion. These findings unveil a novel role for eHsp90 in invasion through the IM and offer valuable mechanistic insights into potential therapeutic approaches for inhibiting Hsp90 to suppress invasion and metastasis.
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