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Creators/Authors contains: "Stevens, Hazel Y."

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  1. Abstract

    Juvenile osteochondritis dissecans (JOCD) is a pediatric disease, which begins with an osteonecrotic lesion in the secondary ossification center which, over time, results in the separation of the necrotic fragment from the parent bone. JOCD predisposes to early-onset osteoarthritis. However, the knowledge gap in JOCD pathomechanisms severely limits current therapeutic strategies. To elucidate its etiology, we conducted a study with induced pluripotent stem cells (iPSCs) from JOCD and control patients. iPSCs from skin biopsies were differentiated to iMSCs (iPSC-derived mesenchymal stromal cells) and subjected to chondrogenic and endochondral ossification, and endoplasmic reticulum (ER)-stress induction assays. Our study, using 3 JOCD donors, showed that JOCD cells have lower chondrogenic capability and their endochondral ossification process differs from control cells; yet, JOCD- and control-cells accomplish osteogenesis of similar quality. Our findings show that endoplasmic reticulum stress sensing and response mechanisms in JOCD cells, which partially regulate chondrocyte and osteoblast differentiation, are related to these differences. We suggest that JOCD cells are more sensitive to ER stress than control cells, and in pathological microenvironments, such as microtrauma and micro-ischemia, JOCD pathogenesis pathways may be initiated. This study is the first, to the best of our knowledge, to realize the important role that resident cells and their differentiating counterparts play in JOCD and to put forth a novel etiological hypothesis that seeks to consolidate and explain previously postulated hypotheses. Furthermore, our results establish well-characterized JOCD-specific iPSC-derived in vitro models and identified potential targets which could be used to improve diagnostic tools and therapeutic strategies in JOCD.

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  2. Mesenchymal stromal cells (MSCs) have been widely investigated for regenerative medicine applications, from treating various inflammatory diseases as a cell therapy to generating engineered tissue constructs. Numerous studies have evaluated the potential effects of MSCs following therapeutic administration. By responding to their surrounding microenvironment, MSCs may mediate immunomodulatory effects through various mechanisms that directly (i.e., contact-dependent) or indirectly (i.e., paracrine activity) alter the physiology of endogenous cells in various disease pathologies. More specifically, a pivotal crosstalk between MSCs and tissue-resident macrophages and monocytes (TMφ) has been elucidated using in vitro and in vivo preclinical studies. An improved understanding of this crosstalk could help elucidate potential mechanisms of action (MOAs) of therapeutically administered MSCs. TMφ, by nature of their remarkable functional plasticity and prevalence within the body, are uniquely positioned as critical modulators of the immune system – not only in maintaining homeostasis but also during pathogenesis. This has prompted further exploration into the cellular and molecular alterations to TMφ mediated by MSCs. In vitro assays and in vivo preclinical trials have identified key interactions mediated by MSCs that polarize the responses of TMφ from a pro-inflammatory (i.e., classical activation) to a more anti-inflammatory/reparative (i.e., alternative activation) phenotype and function. In this review, we describe physiological and pathological TMφ functions in response to various stimuli and discuss the evidence that suggest specific mechanisms through which MSCs may modulate TMφ phenotypes and functions, including paracrine interactions (e.g., secretome and extracellular vesicles), nanotube-mediated intercellular exchange, bioenergetics, and engulfment by macrophages. Continued efforts to elucidate this pivotal crosstalk may offer an improved understanding of the immunomodulatory capacity of MSCs and inform the development and testing of potential MOAs to support the therapeutic use of MSCs and MSC-derived products in various diseases. 
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  3. Abstract

    Large‐scale manufacturing of therapeutic cells requires bioreactor technologies with online feedback control enabled by monitoring of secreted biomolecular critical quality attributes (CQAs). Electrospray ionization mass spectrometry (ESI‐MS) is a highly sensitive label‐free method to detect and identify biomolecules, but requires extensive sample preparation before analysis, making online application of ESI‐MS challenging. We present a microfabricated, monolithically integrated device capable of continuous sample collection, treatment, and direct infusion for ESI‐MS detection of biomolecules in high‐salt solutions. The dynamic mass spectrometry probe (DMSP) uses a microfluidic mass exchanger to rapidly condition samples for online MS analysis by removing interfering salts, while concurrently introducing MS signal enhancers to the sample for sensitive biomolecular detection. Exploiting this active conditioning capability increases MS signal intensity and signal‐to‐noise ratio. As a result, sensitivity for low‐concentration biomolecules is significantly improved, and multiple proteins can be detected from chemically complex samples. Thus, the DMSP has significant potential to serve as an enabling portion of a novel analytical tool for discovery and monitoring of CQAs relevant to therapeutic cell manufacturing.

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