Biomarkers in Cancer Detection, Diagnosis, and Prognosis.

Biomarkers are vital in healthcare as they provide valuable insights into disease diagnosis, prognosis, treatment response, and personalized medicine. They serve as objective indicators, enabling early detection and intervention, leading to improved patient outcomes and reduced costs. Biomarkers also guide treatment decisions by predicting disease outcomes and facilitating individualized treatment plans. They play a role in monitoring disease progression, adjusting treatments, and detecting early signs of recurrence. Furthermore, biomarkers enhance drug development and clinical trials by identifying suitable patients and accelerating the approval process. In this review paper, we described a variety of biomarkers applicable for cancer detection and diagnosis, such as imaging-based diagnosis (CT, SPECT, MRI, and PET), blood-based biomarkers (proteins, genes, mRNA, and peptides), cell imaging-based diagnosis (needle biopsy and CTC), tissue imaging-based diagnosis (IHC), and genetic-based biomarkers (RNAseq, scRNAseq, and spatial transcriptomics).

Multimodal Label-Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers.

Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.

Non-toxic freezing media to retain the stem cell reserves in adipose tissues.

Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) and adipose-derived stromal/stem cells (ASCs) that are inherently multipotent and exhibit regenerative properties. In current practice, lipoaspirate specimens harvested from liposuction surgeries are routinely discarded as a biohazard waste due to a lack of simple, cost effective, and validated cryopreservation protocols. The aim of this study is to develop a xenoprotein-free cryoprotective agent cocktail that will allow for short-term (up to 6 months) preservation of lipoaspirate tissues suitable for fat grafting and/or stromal/stem cell isolation when stored at achievable temperatures (-20 °C or -80 °C). Lipoaspirates donated by three consenting healthy donors undergoing elective cosmetic liposuction surgeries were suspended in five freezing media (FM1: 10% DMSO and 35% BSA; FM2: 2% DMSO and 43% BSA; FM3: 10% DMSO and 35% lipoaspirate saline; FM4: 2% DMSO and 6% HSA; and FM5: 40% lipoaspirate saline and 10% PVP) all suspended in 1X DMEM/F12 and frozen using commercially available freezers (-20 °C or -80 °C) and stored at least for a 1 month. After 1 month of freezing storage, SVF cells and ASCs were isolated from the frozen-thawed lipoaspirates by digestion with collagenase type I. Cell viability was evaluated by fluorescence microscopy after staining with acridine orange and ethidium bromide. The SVF isolated from lipoaspirates frozen at -80 °C retained comparable cell viability with the tested freezing media (FM2, FM3, FM4) comparable with the conventional DMSO and animal serum media (FM1), whereas the FM5 media resulted in lower viability. In contrast, tissues frozen and stored at -20 °C did not yield live SVF cells after thawing and collagenase digestion. The surface marker expression (CD90, CD29, CD34, CD146, CD31, and CD45) of ASCs from frozen lipoaspirates at -80 °C in different cryoprotectant media were also evaluated and no significant differences were found between the groups. The adipogenic and osteogenic differentiation potential were studied by histochemical staining and gene expression by qRT-PCR. Oil Red O staining for adipogenesis revealed that the CPA media FM1, FM4 and FM5 displayed robust differentiation. Alizarin Red S staining for osteogenesis revealed that FM1 and FM4 media displayed superior differentiation in comparison to other tested media. Measurement of adipogenic and osteogenic gene expression by qRT-PCR provided similar outcomes and indicated that FM4 CPA media comparable with FM1 for adipogenesis and osteogenesis.

Single-Cell Analysis Using Hyperspectral Imaging Modalities.

Almost a decade ago, hyperspectral imaging (HSI) was employed by the NASA in satellite imaging applications such as remote sensing technology. This technology has since been extensively used in the exploration of minerals, agricultural purposes, water resources, and urban development needs. Due to recent advancements in optical re-construction and imaging, HSI can now be applied down to micro- and nanometer scales possibly allowing for exquisite control and analysis of single cell to complex biological systems. This short review provides a description of the working principle of the HSI technology and how HSI can be used to assist, substitute, and validate traditional imaging technologies. This is followed by a description of the use of HSI for biological analysis and medical diagnostics with emphasis on single-cell analysis using HSI.

Cryopreserved Adipose Tissue-Derived Stromal/Stem Cells: Potential for Applications in Clinic and Therapy.

Adipose-Derived Stromal/Stem Cells (ASC) have considerable potential for regenerative medicine due to their abilities to proliferate, differentiate into multiple cell lineages, high cell yield, relative ease of acquisition, and almost no ethical concerns since they are derived from adult tissue. Storage of ASC by cryopreservation has been well described that maintains high cell yield and viability, stable immunophenotype, and robust differentiation potential post-thaw. This ability is crucial for banking research and for clinical therapeutic purposes that avoid the morbidity related to repetitive liposuction tissue harvests. ASC secrete various biomolecules such as cytokines which are reported to have immunomodulatory properties and therapeutic potential to reverse symptoms of multiple degenerative diseases/disorders. Nevertheless, safety regarding the use of these cells clinically is still under investigation. This chapter focuses on the different aspects of cryopreserved ASC and the methods to evaluate their functionality for future clinical use.

The Relative Functionality of Freshly Isolated and Cryopreserved Human Adipose-Derived Stromal/Stem Cells.

The capability of multipotent mesenchymal stem cells to maintain cell viability, phenotype and differentiation ability upon thawing is critical if they are to be banked and used for future therapeutic purposes. In the present study, we examined the effect of 9-10 months of cryostorage on the morphology, immunophenotype, colony-forming unit (CFU) and differentiation capacity of fresh and cryopreserved human adipose-derived stromal/stem cells (ASCs) from the same donors. Cryopreservation did not reduce the CFU frequency and the expression levels of CD29, CD73, CD90 and CD105 remained unchanged with the exception of CD34 and CD45; however, the differentiation capacity of cryopreserved ASCs relative to fresh cells was significantly reduced. While our findings suggest that future studies are warranted to improve cryopreservation methods and agents, cryopreserved ASCs retain sufficient features to ensure their practical utility for both research and clinical applications.

A review of biotransport education in the 21st century: lessons learned from experts.

The field of bioengineering is relatively new and complex including multiple disciplines encompassing areas in science and engineering. Efforts including the National Science Foundation (NSF) sponsored Integrative Graduate Education and Research Traineeship (IGERT) and VaNTH Engineering Research Center in Bioengineering Educational Technologies have been made to establish and disseminate knowledge and proven methods for teaching bioengineering concepts. Further, the summer bioengineering conference (SBC), sponsored by the American Society of Mechanical Engineers' (ASME) Bioengineering Division, was established to provide a meeting place for engineering educators and students having common interests in biological systems. Of the many subdisciplines of bioengineering, biotransport is a key subject that has wide applicability to many issues in engineering, biology, medicine, pharmacology, and environmental science, among others. The absence of standard content, guidelines, and texts needed for teaching biotransport courses to students motivated the Biotransport committee of ASME's Bioengineering Division to establish a biotransport education initiative. Biotransport education workshop sessions were conducted during the SBC 2011, 2012, and 2013 as part of this initiative. The workshop sessions included presentations from experienced faculty covering a spectrum of information from general descriptions of undergraduate biotransport courses to very detailed outlines of graduate courses to successful teaching techniques. A list of texts and references available for teaching biotransport courses at undergraduate and graduate levels has been collated and documented based on the workshop presentations. Further, based on individual teaching experiences and methodologies shared by the presenters, it was noted that active learning techniques, including cooperative and collaborative learning, can be useful for teaching undergraduate courses while problem based learning (PBL) can be a beneficial method for graduate courses. The outcomes of the education initiative will help produce students who are knowledgeable in the subject of biotransport, facile in applying biotransport concepts for solving problems in various application areas, and comfortable with their own abilities as life-long learners.

Adult Stem Cells Freezing Processes and Cryopreservation Protocols.

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. Cryopreservation has shown the most promise but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. The purpose of this chapter is to present a general overview of cryopreservation storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage.

Magnetic Control of Nonmagnetic Living Organisms.

Living organisms inspire the design of microrobots, but their functionality is unmatched. Next-generation microrobots aim to leverage the sensing and communication abilities of organisms through magnetic hybridization, attaching magnetic particles to them for external control. However, the protocols used for magnetic hybridization are morphology specific and are not generalizable. We propose an alternative approach that leverages the principles of negative magnetostatics and magnetophoresis to control nonmagnetic organisms with external magnetic fields. To do this, we disperse model organisms in dispersions of Fe3O4 nanoparticles and expose them to either uniform or gradient magnetic fields. In uniform magnetic fields, living organisms align with the field due to external torque, while gradient magnetic fields generate a negative magnetophoretic force, pushing objects away from external magnets. The magnetic fields enable controlling the position and orientation of Caenorhabditis elegans larvae and flagellated bacteria through directional interactions and magnitude. This control is diminished in live spermatozoa and adult C. elegans due to stronger internal biological activity, i.e., force/torque. Our study presents a method for spatiotemporal organization of living organisms without requiring magnetic hybridization, opening the way for the development of controllable living microbiorobots.

Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue.

STUDY QUESTION: Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue? SUMMARY ANSWER: Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol. WHAT IS KNOWN ALREADY: Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. The influence of the vitrification procedure on the cooling rate was evaluated by a computer model. MAIN RESULTS: After vitrification, warming and long-term grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Müllerian hormone (AMH) and growth differentiation factor-9 (GDF-9) immunostaining. Corpora lutea were also observed, evidencing successful ovulation in all the animals. Stromal tissue quality did not appear to be negatively affected by our cryopreservation procedure, as demonstrated by vascularization and proportions of fibrotic areas, which were similar to those found in fresh ungrafted ovarian tissue. LIMITATIONS, REASONS FOR CAUTION: Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies. WIDER IMPLICATIONS OF THE FINDINGS: In addition to encouraging results obtained with our vitrification procedure for non-human ovarian tissue, this study also showed, for the first time, expression of AMH and GDF-9 in ovarian follicles. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4507.10, grant 3.4.590.08 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, and Department of Mechanical Engineering at Louisiana State University (support to Ram Devireddy), and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors has any competing interests to declare.

Three-Dimensional Scaffolds for Bone Tissue Engineering.

Immobilization using external or internal splints is a standard and effective procedure to treat minor skeletal fractures. In the case of major skeletal defects caused by extreme trauma, infectious diseases or tumors, the surgical implantation of a bone graft from external sources is required for a complete cure. Practical disadvantages, such as the risk of immune rejection and infection at the implant site, are high in xenografts and allografts. Currently, an autograft from the iliac crest of a patient is considered the "gold standard" method for treating large-scale skeletal defects. However, this method is not an ideal solution due to its limited availability and significant reports of morbidity in the harvest site (30%) as well as the implanted site (5-35%). Tissue-engineered bone grafts aim to create a mechanically strong, biologically viable and degradable bone graft by combining a three-dimensional porous scaffold with osteoblast or progenitor cells. The materials used for such tissue-engineered bone grafts can be broadly divided into ceramic materials (calcium phosphates) and biocompatible/bioactive synthetic polymers. This review summarizes the types of materials used to make scaffolds for cryo-preservable tissue-engineered bone grafts as well as the distinct methods adopted to create the scaffolds, including traditional scaffold fabrication methods (solvent-casting, gas-foaming, electrospinning, thermally induced phase separation) and more recent fabrication methods (fused deposition molding, stereolithography, selective laser sintering, Inkjet 3D printing, laser-assisted bioprinting and 3D bioprinting). This is followed by a short summation of the current osteochondrogenic models along with the required scaffold mechanical properties for in vivo applications. We then present a few results of the effects of freezing and thawing on the structural and mechanical integrity of PLLA scaffolds prepared by the thermally induced phase separation method and conclude this review article by summarizing the current regulatory requirements for tissue-engineered products.

Surface Plasmon Resonance (SPR) Sensor for Cancer Biomarker Detection.

A biomarker is a physiological observable marker that acts as a stand-in and, in the best-case scenario, forecasts a clinically significant outcome. Diagnostic biomarkers are more convenient and cost-effective than directly measuring the ultimate clinical outcome. Cancer is among the most prominent global health problems and a major cause of morbidity and death globally. Therefore, cancer biomarker assays that are trustworthy, consistent, precise, and verified are desperately needed. Biomarker-based tumor detection holds a lot of promise for improving disease knowledge at the molecular scale and early detection and surveillance. In contrast to conventional approaches, surface plasmon resonance (SPR) allows for the quick and less invasive screening of a variety of circulating indicators, such as circulating tumor DNA (ctDNA), microRNA (miRNA), circulating tumor cells (CTCs), lipids, and proteins. With several advantages, the SPR technique is a particularly beneficial choice for the point-of-care identification of biomarkers. As a result, it enables the timely detection of tumor markers, which could be used to track cancer development and suppress the relapse of malignant tumors. This review emphasizes advancements in SPR biosensing technologies for cancer detection.

New technologies and reagents in lateral flow assay (LFA) designs for enhancing accuracy and sensitivity.

Lateral flow assays (LFAs) are a popular method for quick and affordable diagnostic testing because they are easy to use, portable, and user-friendly. However, LFA design has always faced challenges regarding sensitivity, accuracy, and complexity of the operation. By integrating new technologies and reagents, the sensitivity and accuracy of LFAs can be improved while minimizing the complexity and potential for false positives. Surface enhanced Raman spectroscopy (SERS), photoacoustic techniques, fluorescence resonance energy transfer (FRET), and the integration of smartphones and thermal readers can improve LFA accuracy and sensitivity. To ensure reliable and accurate results, careful assay design and validation, appropriate controls, and optimization of assay conditions are necessary. Continued innovation in LFA technology is crucial to improving the reliability and accuracy of rapid diagnostic testing and expanding its applications to various areas, such as food testing, water quality monitoring, and environmental testing.

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions.

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0-0.8) at various cooling rates (0.5-250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q(IIF)) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q(CW)) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.

Freezing of Solute-Laden Aqueous Solutions: Kinetics of Crystallization and Heat- and Mass-Transfer-Limited Model.

Following an earlier study, we reexamined the latent heat of fusion during freezing at 5 K/min of twelve different pre-nucleated solute-laden aqueous solutions using a Differential Scanning Calorimeter (DSC) and correlated it with the amount of initially dissolved solids or solutes in the solution. In general, a decrease in DSC-measured heat release (in comparison to that of pure water, 335 mJ/mg) was observed with an increasing fraction of dissolved solids or solutes, as observed in the earlier study. In addition, the kinetics of ice crystallization was also obtained in three representative biological media by performing additional experiments at 1, 5 and 20 K/min. A model of ice crystallization based on the phase diagram of a water-NaCl binary solution and a modified Avrami-like model of kinetics was then developed and fit to the experimental data. Concurrently, a heat and mass transfer model of the freezing of a salt solution in a small container is also presented to account for the effect of the cooling rate as well as the solute concentration on the measured latent of freezing. This diffusion-based model of heat and mass transfer was non-dimensionalized, solved using a numerical scheme and compared with experimental results. The simulation results show that the heat and mass transfer model can predict (± 10%) the experimental results.

microRNA Sequencing of CD34+ Sorted Adipose Stem Cells Undergoing Endotheliogenesis.

While several microRNAs (miRNAs) that regulate the endotheliogenesis and further promote angiogenesis have been identified in various cancers, the identification of miRNAs that can drive the differentiation of adipose derived stromal/stem cells (ASCs) into the endothelial lineage has been largely unexplored. In this study, CD34+ ASCs sorted using magnetic bead separation were induced to differentiate along the endothelial pathway. miRNA sequencing of ASCs at day 3, 9, and 14 of endothelial differentiation was performed on Ion Proton sequencing system. The data obtained by this high-throughput method were aligned to the human genome HG38, and the differentially expressed miRNAs during endothelial differentiation at various time points (day 3, 9, and 14) were identified. The gene targets of the identified miRNAs were obtained through miRWalk database. The network-pathway analysis of miRNAs and their targets was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools to determine the potential candidate miRNAs that promote endothelial differentiation. Based on these analyses, six upregulated miRNAs (miR-181a-5p, miR-330-5p, miR-335-3p, miR-15b-5p, miR-99a-5p, and miR-199a-5p) and six downregulated miRNAs (miR-145-5p, miR-155-5p, miR-193a-3p, miR-125a-5p, miR-221-5p, and miR-222-3p) were chosen for further studies. In vitro evaluation of these miRNAs to induce endothelial differentiation when transfected into CD34+ sorted ASCs was studied using Von Willebrand Factor (VWF) staining and quantitative real time-polymerase chain reaction (qRT-PCR). Our results suggest that miRNAs: 335-5p, 330-5p, 181a-5p and anti-miRNAs: 125a-5p, 145-5p can likely induce endothelial differentiation in CD34+ sorted ASCs. Further studies are clearly required to elucidate the specific mechanisms on how miRNAs or anti-miRNAs identified through bioinformatics approach can induce the endotheliogenesis in ASCs.

Dark-field hyperspectral imaging for label free detection of nano-bio-materials.

Nanomaterials are playing an increasingly important role in cancer diagnosis and treatment. Nanoparticle (NP)-based technologies have been utilized for targeted drug delivery during chemotherapies, photodynamic therapy, and immunotherapy. Another active area of research is the toxicity studies of these nanomaterials to understand the cellular uptake and transport of these materials in cells, tissues, and environment. Traditional techniques such as transmission electron microscopy, and mass spectrometry to analyze NP-based cellular transport or toxicity effect are expensive, require extensive sample preparation, and are low-throughput. Dark-field hyperspectral imaging (DF-HSI), an integration of spectroscopy and microscopy/imaging, provides the ability to investigate cellular transport of these NPs and to quantify the distribution of them within bio-materials. DF-HSI also offers versatility in non-invasively monitoring microorganisms, single cell, and proteins. DF-HSI is a low-cost, label-free technique that is minimally invasive and is a viable choice for obtaining high-throughput quantitative molecular analyses. Multimodal imaging modalities such as Fourier transform infrared and Raman spectroscopy are also being integrated with HSI systems to enable chemical imaging of the samples. HSI technology is being applied in surgeries to obtain molecular information about the tissues in real-time. This article provides brief overview of fundamental principles of DF-HSI and its application for nanomaterials, protein-detection, single-cell analysis, microbiology, surgical procedures along with technical challenges and future integrative approach with other imaging and measurement modalities. This article is categorized under: Diagnostic Tools > in vitro Nanoparticle-Based Sensing Diagnostic Tools > in vivo Nanodiagnostics and Imaging Implantable Materials and Surgical Technologies > Nanoscale Tools and Techniques in Surgery.

Breast Cancer Reconstruction: Design Criteria for a Humanized Microphysiological System.

International regulatory agencies such as the Food and Drug Administration have mandated that the scientific community develop humanized microphysiological systems (MPS) as an in vitro alternative to animal models in the near future. While the breast cancer research community has long appreciated the importance of three-dimensional growth dynamics in their experimental models, there are remaining obstacles preventing a full conversion to humanized MPS for drug discovery and pathophysiological studies. This perspective evaluates the current status of human tissue-derived cells and scaffolds as building blocks for an "idealized" breast cancer MPS based on bioengineering design principles. It considers the utility of adipose tissue as a potential source of endothelial, lymphohematopoietic, and stromal cells for the support of breast cancer epithelial cells. The relative merits of potential MPS scaffolds derived from adipose tissue, blood components, and synthetic biomaterials is evaluated relative to the current "gold standard" material, Matrigel, a murine chondrosarcoma-derived basement membrane-enriched hydrogel. The advantages and limitations of a humanized breast cancer MPS are discussed in the context of in-process and destructive read-out assays. Impact statement Regulatory authorities have highlighted microphysiological systems as an emerging tool in breast cancer research. This has been led by calls for more predictive human models and reduced animal experimentation. This perspective describes how human-derived cells, extracellular matrices, and hydrogels will provide the building blocks to create breast cancer models that accurately reflect diversity at multiple levels, that is, patient ethnicity, pathophysiology, and metabolic status.

Statistical thermodynamics of biomembranes.

An overview of the major issues involved in the statistical thermodynamic treatment of phospholipid membranes at the atomistic level is summarized: thermodynamic ensembles, initial configuration (or the physical system being modeled), force field representation as well as the representation of long-range interactions. This is followed by a description of the various ways that the simulated ensembles can be analyzed: area of the lipid, mass density profiles, radial distribution functions (RDFs), water orientation profile, deuterium order parameter, free energy profiles and void (pore) formation; with particular focus on the results obtained from our recent molecular dynamic (MD) simulations of phospholipids interacting with dimethylsulfoxide (Me(2)SO), a commonly used cryoprotective agent (CPA).

Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq.

Adipose-derived stromal/stem cells (ASCs) are multipotent in nature that can be differentiated into various cells lineages such as adipogenic, osteogenic, and chondrogenic. The commitment of a cell to differentiate into a particular lineage is regulated by the interplay between various intracellular pathways and their resultant secretome. Similarly, the interactions of cells with the extracellular matrix (ECM) and the ECM bound growth factors instigate several signal transducing events that ultimately determine ASC differentiation. In this study, RNA-sequencing (RNA-Seq) was performed to identify the transcriptome profile of osteogenic induced ASCs to understand the associated genotype changes. Gene ontology (GO) functional annotations analysis using Database for Annotation Visualization and Integrated Discovery (DAVID) bioinformatics resources on the differentially expressed genes demonstrated the enrichment of pathways mainly associated with ECM organization and angiogenesis. We, therefore, studied the expression of genes coding for matrisome proteins (glycoproteins, collagens, proteoglycans, ECM-affiliated, regulators, and secreted factors) and ECM remodeling enzymes (MMPs, integrins, ADAMTSs) and the expression of angiogenic markers during the osteogenesis of ASCs. The upregulation of several pro-angiogenic ELR+ chemokines and other angiogenic inducers during osteogenesis indicates the potential role of the secretome from differentiating ASCs in the vascular development and its integration with the bone tissue. Furthermore, the increased expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 involved in the WNT, TGF-ß, JNK, HedgeHog and ERK1/2 pathways suggests the regulation of osteogenesis through interplay between these pathways. The RNA-Seq data was also validated by performing QPCR on selected up- and down-regulated genes (COL10A1, COL11A1, FBLN, FERMT1, FN1, FOXF1, LAMA3, LAMA4, LAMB1, IGF1, WNT10B, MMP1, MMP3, MMP16, ADAMTS6, and ADAMTS14).