The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of ...development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.
The only definitive treatment for end-stage organ failure is orthotopic transplantation. Lung extracellular matrix (LECM) holds great potential as a scaffold for lung tissue engineering because it ...retains the complex architecture, biomechanics, and topologic specificity of the lung. Decellularization of human lungs rejected from transplantation could provide "ideal" biologic scaffolds for lung tissue engineering, but the availability of such lungs remains limited. The present study was designed to determine whether porcine lung could serve as a suitable substitute for human lung to study tissue engineering therapies.
Human and porcine lungs were procured, sliced into sheets, and decellularized by three different methods. Compositional, ultrastructural, and biomechanical changes to the LECM were characterized. The suitability of LECM for cellular repopulation was evaluated by assessing the viability, growth, and metabolic activity of human lung fibroblasts, human small airway epithelial cells, and human adipose-derived mesenchymal stem cells over a period of 7 days.
Decellularization with 3-(3-Cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS) showed the best maintenance of both human and porcine LECM, with similar retention of LECM proteins except for elastin. Human and porcine LECM supported the cultivation of pulmonary cells in a similar way, except that the human LECM was stiffer and resulted in higher metabolic activity of the cells than porcine LECM.
Porcine lungs can be decellularized with CHAPS to produce LECM scaffolds with properties resembling those of human lungs, for pulmonary tissue engineering. We propose that porcine LECM can be an excellent screening platform for the envisioned human tissue engineering applications of decellularized lungs.
Over the past 15 years, mesenchymal stem cells (MSCs) have been assessed for their capacity to suppress inflammation and promote tissue repair. Regardless of whether the cells are primed (exposed to ...instructive cues) before administration, their phenotype will respond to environmental signals present in the pathophysiological setting being treated. Since hypoxia and inflammation coexist in the settings of acute injury and chronic disease we sought to explore how the proteome and metabolome of MSCs changes when cells were exposed to 48 h of 1% oxygen, interferon gamma (IFN-γ), or both cues together. We specifically focused on changes in cell metabolism, immune modulation, extracellular matrix secretion and modification, and survival capacity. IFN-γ promoted expression of anti-pathogenic proteins and induced MSCs to limit inflammation and fibrosis while promoting their own survival. Hypoxia instead led to cell adaptation to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. While dual priming resulted in additive effects, we also found many instances of synergy. These data lend insight to how MSCs may behave after administration to a patient and suggest how priming cells beforehand could improve their therapeutic capacity.
The number of available donor organs limits lung transplantation, the only lifesaving therapy for the increasing population of patients with end-stage lung disease. A prevalent etiology of injury ...that renders lungs unacceptable for transplantation is gastric aspiration, a deleterious insult to the pulmonary epithelium. Currently, severely damaged donor lungs cannot be salvaged with existing devices or methods. Here we report the regeneration of severely damaged lungs repaired to meet transplantation criteria by utilizing an interventional cross-circulation platform in a clinically relevant swine model of gastric aspiration injury. Enabled by cross-circulation with a living swine, prolonged extracorporeal support of damaged lungs results in significant improvements in lung function, cellular regeneration, and the development of diagnostic tools for non-invasive organ evaluation and repair. We therefore propose that the use of an interventional cross-circulation platform could enable recovery of otherwise unsalvageable lungs and thus expand the donor organ pool.
One of the oldest mechanisms of immune defense against pathogens is through detection of foreign DNA. Since human DNA is compartmentalized into the nucleus, its presence in the cytosol heralds a ...potential threat. The cGAS-STING pathway is one of the most important cytosolic DNA sensing pathways and leads to interferon signaling, inflammasome activation, autophagy, and cell death. While STING signaling is protective at physiologic levels, chronic activation of this pathway can instead drive autoinflammation and autoimmunity. Here we discuss several monogenic disorders of the STING pathway that highlight its impact on both innate and adaptive immunity in the progressive loss of tolerance. The potential relevance of STING signaling in systemic lupus erythematosus is then discussed with a focus on future avenues for monitoring and targeting this pathway.
Mesenchymal stromal cells (MSCs) are a promising cell source for promoting tissue repair, due to their ability to release growth, angiogenic, and immunomodulatory factors. However, when injected as a ...suspension, these cells suffer from poor survival and localization, and suboptimal release of paracrine factors. While there have been attempts to overcome these limitations by modifying MSCs themselves, a more versatile solution is to grow them in three dimensions, as aggregates or embedded into biomaterials. Here we review the mechanisms by which 3D culture can influence the regenerative capacity of undifferentiated MSCs, focusing on recent examples from the literature. We further discuss how knowledge of these mechanisms can lead to strategic design of MSC therapies that overcome some of the challenges to their effective translation.
BACKGROUNDMacrophage activation syndrome (MAS) is a life-threatening complication of Still's disease (SD) characterized by overt immune cell activation and cytokine storm. We aimed to further ...understand the immunologic landscape of SD and MAS.METHODWe profiled PBMCs from people in a healthy control group and patients with SD with or without MAS using bulk RNA-Seq and single-cell RNA-Seq (scRNA-Seq). We validated and expanded the findings by mass cytometry, flow cytometry, and in vitro studies.RESULTSBulk RNA-Seq of PBMCs from patients with SD-associated MAS revealed strong expression of genes associated with type I interferon (IFN-I) signaling and cell proliferation, in addition to the expected IFN-γ signal, compared with people in the healthy control group and patients with SD without MAS. scRNA-Seq analysis of more than 65,000 total PBMCs confirmed IFN-I and IFN-γ signatures and localized the cell proliferation signature to cycling CD38+HLA-DR+ cells within CD4+ T cell, CD8+ T cell, and NK cell populations. CD38+HLA-DR+ lymphocytes exhibited prominent IFN-γ production, glycolysis, and mTOR signaling. Cell-cell interaction modeling suggested a network linking CD38+HLA-DR+ lymphocytes with monocytes through IFN-γ signaling. Notably, the expansion of CD38+HLA-DR+ lymphocytes in MAS was greater than in other systemic inflammatory conditions in children. In vitro stimulation of PBMCs demonstrated that IFN-I and IL-15 - both elevated in MAS patients - synergistically augmented the generation of CD38+HLA-DR+ lymphocytes, while Janus kinase inhibition mitigated this response.CONCLUSIONMAS associated with SD is characterized by overproduction of IFN-I, which may act in synergy with IL-15 to generate CD38+HLA-DR+ cycling lymphocytes that produce IFN-γ.
Regulatory T cells (Tregs) are critical for enforcing peripheral tolerance. Monogenic "Tregopathies" affecting Treg development, stability, and/or function commonly present with polyautoimmunity, ...atopic disease, and infection. While autoimmune manifestations may present in early childhood, as more disorders are characterized, conditions with later onset have been identified. Treg numbers in the blood may be decreased in Tregopathies, but this is not always the case, and genetic testing should be pursued when there is high clinical suspicion. Currently, hematopoietic cell transplantation is the only curative treatment, but gene therapies are in development, and small molecule inhibitors/biologics may also be used.
Chronic recurrent multifocal osteomyelitis (CRMO) is an uncommon cause of chronic inflammatory bone pain in children that can be disabling. Often, this diagnosis is considered only after a prolonged ...workup, leading to frustration for families and unnecessary interventions for patients. Here we describe three cases of CRMO to increase awareness of how it may present. The first patient had a typical presentation of focal bone pain (knee), for which she underwent bone scan (hint of >1 lesion), had a bone biopsy to rule out malignancy, received empiric antibiotics for presumed infection, and finally had whole-body imaging confirming CRMO when symptoms persisted. The second patient had a similar workup, but initially presented with clavicular pain. This location should raise suspicion for CRMO, as it is an uncommon location for infectious osteomyelitis. The third patient presented with delayed growth and right hip pain, and simultaneously developed palmoplantar pustulosis. These secondary findings can also serve as red flags for CRMO, as it has been linked to this skin condition and inflammatory bowel disease. All patients improved on non-steroidal anti-inflammatory (NSAID) medications, methotrexate, and/or tumor necrosis factor (TNF)-α antagonists. By raising awareness of clinical findings suggestive of CRMO, this report may help expedite diagnosis, so patients can be started on anti-inflammatory therapy.Chronic recurrent multifocal osteomyelitis (CRMO) is an uncommon cause of chronic inflammatory bone pain in children that can be disabling. Often, this diagnosis is considered only after a prolonged workup, leading to frustration for families and unnecessary interventions for patients. Here we describe three cases of CRMO to increase awareness of how it may present. The first patient had a typical presentation of focal bone pain (knee), for which she underwent bone scan (hint of >1 lesion), had a bone biopsy to rule out malignancy, received empiric antibiotics for presumed infection, and finally had whole-body imaging confirming CRMO when symptoms persisted. The second patient had a similar workup, but initially presented with clavicular pain. This location should raise suspicion for CRMO, as it is an uncommon location for infectious osteomyelitis. The third patient presented with delayed growth and right hip pain, and simultaneously developed palmoplantar pustulosis. These secondary findings can also serve as red flags for CRMO, as it has been linked to this skin condition and inflammatory bowel disease. All patients improved on non-steroidal anti-inflammatory (NSAID) medications, methotrexate, and/or tumor necrosis factor (TNF)-α antagonists. By raising awareness of clinical findings suggestive of CRMO, this report may help expedite diagnosis, so patients can be started on anti-inflammatory therapy.
End-stage lung disease is the third leading cause of death worldwide, accounting for 400,000 deaths per year in the United States alone. To reduce the morbidity and mortality associated with lung ...disease, new therapeutic strategies aimed at promoting lung repair and increasing the number of donor lungs available for transplantation are being explored. Because of the extreme complexity of this organ, previous attempts at bioengineering functional lungs from fully decellularized or synthetic scaffolds lacking functional vasculature have been largely unsuccessful. An intact vascular network is critical not only for maintaining the blood-gas barrier and allowing for proper graft function but also for supporting the regenerative cells. We therefore developed an airway-specific approach to removing the pulmonary epithelium, while maintaining the viability and function of the vascular endothelium, using a rat model. The resulting vascularized lung grafts supported the attachment and growth of human adult pulmonary cells and stem cell-derived lung-specified epithelial cells. We propose that de-epithelialization of the lung with preservation of intact vasculature could facilitate cell therapy of pulmonary epithelium and enable bioengineering of functional lungs for transplantation.
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