Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

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The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.

/pdf/characterization-of-perinatal-stem-cell-spheroids-for-the-31aof575.pdf

Characterization of Perinatal Stem Cell Spheroids for the Development of Cell Therapy Strategy

When students repeatedly reflect, it can enhance their metacognitive abilities, including self-regulatory skills of planning, monitoring, and evaluating. In a fluid mechanics course for undergraduates at a large southeastern U.S. university, in-class problem solving in a flipped classroom was coupled with intentional metacognitive skills instruction and repeated reflection to enhance metacognition. The weekly reflective responses were coded by two analysts to identify the recurring themes and uncover evidence of the development and/or reinforcement of self-regulating behaviors for academic management. To enable a comparison, a flipped classroom without the metacognitive instruction and repeated reflection was also implemented (i.e., non-intervention group). The two cohorts completed identical final exams. Based on our preliminary analysis with year one data, a statistically and practically-significant difference between the two cohorts was found with the free-response scores on the final exam in favor of the intervention cohort that had received the metacognitive support ( p < 0.0005; Cohen's d = 0.72). Also, the Metacognitive Activities Inventory (MCAI) indicated a significantly-higher positive change in self-regulatory behavior for the intervention cohort ( p = 0.001; d = 0.50). Focus groups were conducted to gather students’ perspectives on the reflective activity, with differences found by demographic group. In addition, a significantly higher proportion of females (versus males) viewed the reflections in a positive manner ( p = 0.05). Significant associations between themes in the weekly reflections and direct knowledge measures were also uncovered. This included a positive relationship between academic self-management (i.e., diligence and carefulness) and exam performance. Overall, our preliminary results point to a desirable impact of metacognitive instruction and repeated reflection on knowledge outcomes, metacognitive skills, and self-regulatory behaviors.

Metacognition instruction and repeated reflection in a fluid mechanics course: Reflective themes and student outcomes

Background: Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration. Methods: We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro. Results: The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield. Conclusions: A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology.

https://www.mdpi.com/2073-4409/12/13/1793/pdf?version=1688629263

Adult Multipotent Cardiac Progenitor-Derived Spheroids: A Reproducible Model of In Vitro Cardiomyocyte Commitment and Specification

Abstract Mesenchymal stromal cells (MSCs) are under investigation for wound healing and tissue regeneration due to their potent secretome. Compared to monodisperse cells, MSC spheroids exhibit increased cell survival and enhanced secretion of endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE 2 ), two key factors in wound repair. We previously upregulated the proangiogenic potential of homotypic MSC spheroids by manipulating microenvironmental culture conditions. However, this approach depends on the responsiveness of host endothelial cells (ECs)—a limitation when attempting to restore large tissue deficits and for patients with chronic wounds in which ECs are dysfunctional and unresponsive. To address this challenge, we used a Design of Experiments (DOE) approach to engineer functionally distinct MSC spheroids that maximize VEGF production (VEGF MAX ) or PGE 2 production (PGE 2,MAX ) while incorporating ECs that could serve as the basic building blocks for vessel formation. VEGF MAX produced 22.7-fold more VEGF with enhanced endothelial cell migration compared to PGE 2,MAX , while PGE 2,MAX produced 16.7-fold more PGE 2 with accelerated keratinocyte migration compared to VEGF MAX . When encapsulated together in engineered protease-degradable hydrogels as a model of cell delivery, VEGF MAX and PGE 2,MAX spheroids exhibited robust spreading into the biomaterial and enhanced metabolic activity. The distinct bioactivities of these MSC spheroids demonstrate the highly tunable nature of spheroids and provide a new approach to leverage the therapeutic potential of cell-based therapies. 

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Tuning the Microenvironment to Create Functionally Distinct Mesenchymal Stromal Cell Spheroids

Cell therapies offer a tailorable, personalized treatment for use in tissue engineering to address defects arising from trauma, inefficient wound repair, or congenital malformation. However, most cell therapies have achieved limited success to date. Typically injected in solution as monodispersed cells, transplanted cells exhibit rapid cell death or insufficient retention at the site, thereby limiting their intended effects to only a few days. Spheroids, which are dense, three-dimensional (3D) aggregates of cells, enhance the beneficial effects of cell therapies by increasing and prolonging cell-cell and cell-matrix signaling. The use of spheroids is currently under investigation for many cell types. Among cells under evaluation, spheroids formed of mesenchymal stromal cells (MSCs) are particularly promising. MSC spheroids not only exhibit increased cell survival and retained differentiation, but they also secrete a potent secretome that promotes angiogenesis, reduces inflammation, and attracts endogenous host cells to promote tissue regeneration and repair. However, the clinical translation of spheroids has lagged behind promising preclinical outcomes due to hurdles in their formation, instruction, and use that have yet to be overcome. This review will describe the current state of preclinical spheroid research and highlight two key examples of spheroid use in clinically relevant disease modeling. It will highlight techniques used to instruct the phenotype and function of spheroids, describe current limitations to their use, and offer suggestions for the effective translation of cell spheroids for therapeutic treatments. 

/pdf/strategies-to-capitalize-on-cell-spheroid-therapeutic-4ul23j3q.pdf

Strategies to capitalize on cell spheroid therapeutic potential for tissue repair and disease modeling

Metacognitive skills can have enormous benefits for students within engineering courses. Unfortunately, these metacognitive skills tend to fall outside the content area of most courses, and consequently, they can often be neglected in instruction. In this context, previous research on concept mapping as a teaching strategy points to meaningful learning. The purpose of this innovation paper is to report an application of concept mapping (1) to facilitate metacognition steps in students, and (2) to identify the muddiest points students struggle with, during both in-person and online instruction of a problem-solving-based biomedical engineering course. This innovation article also looks at the usefulness of concept mapping through instructor and student perceptions and students' class performance. The entire concept mapping intervention was conducted during weeks 8-10 of the Spring 2019 in-person quarter and during weeks 3-4 and 8-10 of the Spring 2021 online quarter. The exercise involved concept mapping, explanation and discussion with peers, and answering structured reflection prompts. Each concept map activity was contextualized to the metacognitive knowledge domain of the revised Bloom's taxonomy. The average class performance was compared between students who completed concept mapping vs. those who did not, using a t-test and one-way ANOVA at alpha = 0.05 significance level followed by a Tukey HSD test. Students' concept maps and reported answers were analyzed qualitatively following the concept mapping intervention. During the Spring 2019 in-person quarter, 59.30% of students completed concept mapping with reflection, whereas 47.67% completed it in spring 2021 online instruction. A two-tailed, unpaired t-test indicated that concept mapping did not significantly enhance students' class performance (p > 0.05) within each of the in-person and online instructions. Peers' suggestions to students to improve concept maps revealed themes related to course concepts, prerequisite concepts, and the act of concept mapping itself. Concept mapping was effective in revealing the muddiest points of the course. Concept mapping did not significantly enhance students' class performance either in-person or online instruction (effect sizes were 0.29 for the 2019 in-person quarter and 0.33 for the 2021 online quarter). However, instructors and students' perceptions reflected that concept mapping facilitated metacognition in a problem-solving-based biomedical engineering course both during in-person and online instruction. Most students (78%) were optimistic about the usefulness of concept mapping for this course, and 84% were inclined to apply it for a variety of other courses.The online version contains supplementary material available at 10.1007/s43683-022-00066-3. 

/pdf/concept-mapping-as-a-metacognition-tool-in-a-problem-solving-124l4dkf.pdf

Concept Mapping as a Metacognition Tool in a Problem-Solving-Based BME Course During In-Person and Online Instruction

Despite recent technological advances such as ex vivo lung perfusion (EVLP), the outcome of lung transplantation remains unsatisfactory with ischemic injury being a common cause for primary graft dysfunction. New therapeutic developments are hampered by limited understanding of pathogenic mediators of ischemic injury to donor lung grafts. Here, to identify novel proteomic effectors underlying the development of lung graft dysfunction, using bioorthogonal protein engineering, we selectively captured and identified Newly Synthesized glycoproteins (NewS-glycoprotein) produced during EVLP with unprecedented temporal resolution of 4 hours. Comparing the NewS-glycoproteomes in lungs with and without warm ischemia injury, we discovered highly specific proteomic signatures with altered synthesis in ischemic lungs, which exhibited close association to hypoxia response pathways. Inspired by the discovered protein signatures, pharmacological modulation of the calcineurin pathway during EVLP of ischemic lungs offered graft protection and improved post-transplantation outcome. In summary, the described EVLP-NewS-glycoproteomics strategy delivers an effective new means to reveal molecular mediators of donor lung pathophysiology and offers the potential to guide future therapeutic development.

Newly-synthesized Glycoprotein Profiling to Identify Molecular Signatures of Warm Ischemic Injury in Donor Lungs.

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner.

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Shierly W Fok

Papers

Strategies to capitalize on cell spheroid therapeutic potential for tissue repair and disease modeling

Concept Mapping as a Metacognition Tool in a Problem-Solving-Based BME Course During In-Person and Online Instruction

Newly-synthesized Glycoprotein Profiling to Identify Molecular Signatures of Warm Ischemic Injury in Donor Lungs.

Macromolecular crowding and decellularization method increase the growth factor binding potential of cell-secreted extracellular matrices