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The favorable biological and mechanical properties of the most common components of the placenta, the amnion and chorion, have been explored for regenerative medical indications. The use of the combination of amnion and chorion has also become very popular. But, published data from placental tissues in their final, useable form is lacking. During treatment with membrane product, the tissue is usually sterile, intact and laid on a wound or treatment area. The factors available to the treatment area from the applied product need to be elucidated and presented in a relatable form. Current reporting for eluted growth factor results are typically expressed per milliliter, which is not informative with respect to the area of tissue covered by the actual membrane and may differ among techniques.To address this inconsistency, amnion or amnion/chorion were isolated from human placentas and processed by a proprietary procedure. The final dry, sterilized product was evaluated for structural components and growth factor elution. Growth factors were quantified by multiplex panels and ELISAs and the values normalized to specific area and elution volume of finished product. This information allows extrapolation to all membrane sizes and affords cross-study comparisons.Analysis of membrane supernatants show that dehydrated, sterilized amnion and amnion/chorion elute factors that are conducive to wound healing, which are available to recipient tissues. Importantly, these measurable factors eluted from dehydrated, sterilized membranes can be reported as a function of available factors per square centimeter of tissue.The standardized characterization of dehydrated, sterilized amnion and amnion/chorion as delivered to recipient tissues permits understanding and comparison of the products across various graft sizes, types, and eluate volumes. Further, reporting this data as a function of cm

Exosomes are very small extracellular vesicles secreted by cells to local and distant tissues. These mini signal transporters elicit acute and chronic effects on recipient cells. Studies regarding exosomes and their relationship to disease, as well as healthy functions, are eliciting extraordinary excitement as data pours in from groups around the world. Reporting of exosome biogenesis, selective loading of cargo, directed release, and resulting changes in adjacent and distal cells are providing information that is changing the way we view cancer progression and treatment. As a result, the properties of exosomes are being exploited for diagnostic, prognostic, and therapeutic applications. First, by referring to the signaling molecules carried by exosomes, they are being tested as indicators of the presence of transformed cells in early stages of cancer. Secondly, the cargo of exosomes secreted from tumors have been linked to prognostic factors and metastatic properties. Thirdly, exosome-based therapies are being developed which utilize the inherent properties of these mini-transporters to affect and interfere with cancer. Exosome creation, loading, and release plays an important role in cancer formation, progression and organotropic metastasis. The developed and developing therapies should be considered with understanding of their advantages and pitfalls, as well as the various roles exosomes play in normal and pathogenic processes. The combination of previously discovered attributes of exosomes with new discoveries occurring daily provide valuable and additive relevant factors to be considered as we embark on the continued discovery of exosomes and their relationship to cancer diagnostics and therapeutics.

Sarcomas are rare malignancies of mesenchymal origin for which current treatment options are very limited. Due to the vast heterogeneity of the various subtypes, the development of new targeted therapies requires a customized approach. Checkpoint blockade therapies are a promising alternative that still have not been systematically investigated for sarcoma patients. Thus, utilizing the infrastructure of the Cell Therapy Core Facility (CTC) at Nova Southeastern University (NSU), we developed a clinic-to-bench pipeline that enables the processing of fresh tumor material after surgical excision to generate primary sarcoma cell lines for biobanking and research purposes. Using this pipeline, we have extensively characterized mRNA, miRNA and cell surface expression profiles from more than 40 sarcoma cell lines to date, as well as validated their tumorigenicity by assessing expression of sarcoma/cancer associated genes such as Birc5, Birc2, Bcl2, Ewsr1-fli1, Syt/Ssx and Pcna. Moreover, we assessed the expression of immunomodulatory molecules that may suppress antitumor responses, such as PD-L1, PD-L2, OX40L and CD40L, using multicolor flow cytometry. Detailed characterization of the immune profile of 7 bone-related and 7 soft tissue sarcoma cell lines, assessed after 12 weeks of in vitro serial passaging, revealed a common signature in the cell surface expression of proliferative marker PCNA, and lymphocyte ligands CD112 and CD115. Moreover, immunoprofiling of the PCNA/CD112/CD155+ sarcoma cell lines (n=14) demonstrated persistent expression of PD-L2 in all cell lines and the potential applications of currently clinically approved anti-PD-1 checkpoint inhibitors for sarcoma treatment. Importantly, the conserved expression of these proteins enabled the distinction of sarcoma tumor cells from other cells in the fresh primary tumor mix directly after isolation. Thus, we next performed a comparative analysis of the immune profile of 6 freshly isolated sarcoma tumors and their respective cell lines. Comparison of the surface expression profiles of the generated cell lines and the corresponding primary tumor cells showed that the initial phenotype was preserved after more than 12 weeks of expansion and after cryopreservation. This proves that the generated cell lines are an accurate tool that can be used for the development of novel personalized immunotherapies. Most importantly, our results enabled the design of a translational research pipeline that allows the systematic characterization of the immune profile of understudied sarcomas for the first time in order to gain insight into which patients may directly benefit from available immunotherapies and which may be eligible to enroll in clinical trials assessing the efficacy of checkpoint inhibitors. In summary, the generated sarcoma biobank serves as a unique and highly representative primary resource that can be utilized to identify potential candidates for novel targeted and personalized immunotherapies for the treatment of sarcomas. Citation Format: Anna-Maria Georgoudaki, Robin Krueger, Michelle Hartman, Tamara Chinn, Dustin Tran, Reneé Potens, Wendy Weston, H. Thomas Temple, Adil D. Duru. Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A23.

Throughout the history of medical knowledge, either defined by written text or interpreted in archeological discovery, humans have engendered deep respect for disease and the assurance that declining health will lead to death. Recognizing termination is arguably the first step in seeking strategies to prolong life, and the first pause is likely “why.” Along the 5000-year timeline of historical interpretation, ancient civilizations leaned into the convections of spirits as causal to the explicit infliction, assigning the humors, spirits, gods, curses, etc. as the root of the illness. As information evolved, the focal point of understanding cause probably emerged with what we might call “Hippocratic medicine,” which is essentially the belief that medicine should be practiced as a scientific discipline based on the natural sciences, and that diagnosing and preventing diseases as well as treating them can be tied to seeking remedy and restitution of health, youth, and at some level immortality. Extended immortality is a paradox that belies the assurance that life in a single consciousness will end. From contemporary literature, both scientific and lay readers have been exposed to contexts of modern biology where extensions of life beyond that consciousness are now commonly accepted (Skloot, 2010 [1] ). HeLa cells were vital for developing the polio vaccine; uncovered secrets of cancer and viruses; helped lead to in vitro fertilization, cloning, and gene mapping; and have been bought and sold by the billions, yet Henrietta Lacks has been buried for more than 60 years. Do these cells in themselves offer a constancy of immortality? The argument of passing genetic information forward in cell division is clearly a regenerative event, but what level of transfer is necessary for regenerative repair that does not dilute the individual being treated. Examples of blood transfusion, organ transplant, and integration of stem cells are now everyday occurrences. These examples raise a question that currently defies scientific explanation: does chimerism at the organ level, tissue level, or even the cell level bestow varying and inextricable states of awareness, being, and consciousness? In that context, what contribution of genetic exchange is sufficient to constitute hybrid viability…a single base pair, two, five, ten, and how much RNA or DNA is necessary to qualify as a regenerative event? Technology has advanced to what many have accepted as the nanofrontier, and the conversations of nanomedicine abound. From nanodiagnostics through nanocomposites, therapeutic initiatives have sought to identify and resolve assets of understanding that may underpin new techniques of regeneration. As technology advances and perception emerges, defining packaged genetic material in membrane-shrouded entities known as exosomes, what once was considered cell biology may evolve in a subcellular specialization of extracellular phenotypic enrichment. Evidence already exists that exosomes are readily exchanged between cells, and that receipt of these packages endows new constitution and performance. Defining RNA in the context of exosomes couples another level of nanoconsideration; its 0.34 nm per base pair and 10 base pairs per turn qualifies in size if not in function. A possibility to utilize RNA as a vector seems certain. Nature adapted this strategy billions of years ago as an option to decrease cell energy demands, and it remains today as a means of lateral transfer of genetic material without cell division, essentially a component of paracrine activity widely discussed but still not completely understood. Application diversity is pervasive, as applications for gain of function include intervertebral disc and pancreas exocrine systems among others. Use of exosome fractions as an adjuvant to immuno-oncology is similarly caught in this tailwind of discovery. The accent of immortality, specifically which cell lineages might best comport a spectral potential providing a meaningful library of staged development; i.e., cells that accommodate organ coalescence based on exosome expression during morphogenesis rather than propagation of differentiated lineages that the proverbial choir singing in unison are momentarily reflected on. Embryonic cells (EMCs) are politically charged but of course support sufficient pluripotency to populate each and all of the germ layers. However, if one is not seeking the slalom of political flags as the main event of research, other options are available. In contrast to EMCs, most stem cells that have been well characterized are multipotent and can be induced to differentiate into derivatives of two of the three germ layers under appropriate conditions. It is critical to accept the three basic properties that have been ascribed to stem cells. First, self-renewal; stem cells divide to produce identical daughter cells and thereby maintain the stem cell population. Second, stem cells are able to divide asymmetrically to yield an identical cell and a daughter cell that acquires specific morphology, phenotype, and physiological properties that categorize it as a cell belonging to a particular tissue. The third property of stem cells is that they may renew the tissues that they populate. While the trumpets have resounded the age of stem cells, there is growing support that phenotypic expression can occur via lateral genetic transfer in secretion in the form of liposomal inclusions, membrane inclusions, and exosome infusion. This chapter explores umbilical cord stem cells, or cord blood, and addresses potential for modulation in directing durable phenotypic derivatives for regenerative applications.

Aplastic anemia (AA) is an immune-mediated and life-threatening form of acquired bone marrow failure (BMF), characterized by development and expansion of self-reactive T cells. These T cells cause continuous destruction of hematopoietic stem cells (HSCs), progenitors, and mature blood cells, leading to severe and if left untreated fatal marrow hypoplasia and pancytopenia. Standard treatment options for patients with AA include: (1) immunosuppressive therapy (IST) with anti-thymocyte globulin and cyclosporine A which targets self-reactive T cells, or (2) matched sibling or unrelated BM transplant (BMT). The IST treatment is often not effective due to poor response to therapy or disease relapse after IST. Also, BMT is not an option for many patients due to their age, comorbidities, and the lack of histocompatible donor. This necessitates development and testing of novel approaches to reduce severity of AA and to efficiently treat patients with refractory and relapsed AA. Immune-mediated AA was reproduced in animals, including mouse lymphocyte infusion models, which are used to study further etiology and pathophysiology of AA and test new drugs and approaches in treating and managing AA. In these mouse models the immune correlates and pathologic features of AA are strikingly similar to features of severe human AA. In this article we (a) briefly review standard and developing approaches for treating AA and (b) describe development and testing of novel treatment approach with a potential to safely reduce BM hypoplasia and significantly decrease the loss of HSCs in mouse lymphocyte infusion model of AA.

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.