Adipose tissue dysfunction is critical to the development of type II diabetes and other metabolic diseases. While monolayer cell culture has been useful for studying fat biology, 2D culture often does not reflect the complexity of fat tissue. Animal models are also problematic in that they are expensive, time consuming, and may not completely recapitulate human biology because of species variation. To address these problems, we have developed a scaffold-free method to generate 3D adipose spheroids from primary or immortal human or mouse pre-adipocytes. Pre-adipocytes self-organize into spheroids in hanging drops and upon transfer to low attachment plates, can be maintained in long-term cultures. Upon exposure to differentiation cues, the cells mature into adipocytes, accumulating large lipid droplets that expand with time. The 3D spheroids express and secrete higher levels of adiponectin compared to 2D culture and respond to stress, either culture-related or toxin-associated, by secreting pro-inflammatory adipokines. In addition, 3D spheroids derived from brown adipose tissue (BAT) retain expression of BAT markers better than 2D cultures derived from the same tissue. Thus, this model can be used to study both the maturation of pre-adipocytes or the function of mature adipocytes in a 3D culture environment.

The ability to use mesenchymal stromal cells (MSC) directly out of cryostorage would significantly reduce the logistics of MSC therapy by allowing on-site cryostorage of therapeutic doses of MSC at hospitals and clinics. Such a paradigm would be especially advantageous for the treatment of acute conditions such as stroke and myocardial infarction, which are likely to require treatment within hours after ischemic onset. Recently, several reports have emerged that suggest MSC viability and potency are damaged by cryopreservation. Herein we examine the effect of cryopreservation on human MSC viability, immunomodulatory potency, growth factor secretion, and performance in an ischemia/ reperfusion injury model. Using modifications of established cryopreservation methods we developed MSC that retain >95% viability upon thawing, remain responsive to inflammatory signals, and are able to suppress activated human peripheral blood mononuclear cells. Most importantly, when injected into the eyes of mice 3 hours after the onset of ischemia and 2 hours after the onset of reperfusion, cryopreserved performed as well as fresh MSC to rescue retinal ganglion cells. Thus, our data suggests when viability is maintained throughout the cryopreservation process, MSC retain their therapeutic potency in both in vitro potency assays and an in vivo ischemia/reperfusion model.Mesenchymal stromal/stem cells (MSC) have been explored in hundreds of clinical trials for the treatment of dozens of conditions 1,2 . While MSC can be harvested from nearly any tissue 3 , they are a rare cell type 4 and thus typically require significant ex vivo expansion to generate therapeutic doses of cells. Allogeneic MSC are used in most clinical trials as MSC are immune evasive, allowing them to avoid immediate immune detection and clearance 2 . Allogeneic MSC are typically expanded in culture, cryopreserved, and banked for future use, creating the opportunity for an 'off-the-shelf ' therapy.Many proposed applications of MSC therapy would require on demand access to therapeutic doses of MSC and therefore necessitate access to cryopreserved MSC stocks. Acute conditions including acute graft versus host disease (GvHD), acute kidney injury, acute lung injury, and sudden onset ischemic events such as myocardial infarction, acute limb ischemia, retinal and optic neuropathies, and stroke would all benefit from rapid MSC administration within hours after the onset of symptoms. The mechanism of action of MSC in these conditions is thought to be mediated through both modulation of inflammatory reactions as well as secretion of protective growth factors 5 . Even if a disease indication could accommodate a post-thaw recovery period ranging from hours to days, logistically, use of MSC immediately post-thaw would still be preferable, since post-thaw recovery needs to be carried out by experienced technicians in dedicated facilities. This not only leads to quality control issues but also adds significant infrastructure requirements that will prevent the use of MSC therapies...

The use of mesenchymal stromal cell (MSC) therapy for the treatment of type 2 diabetes (T2D) and T2D complications is promising; however, the investigation of MSC function in the setting of T2D has not been thoroughly explored. In our current study, we investigated the phenotype and function of MSCs in a simulated in vitro T2D environment. We show that palmitate, but not glucose, exposure impairs MSC metabolic activity with moderate increases in apoptosis, while drastically affecting proliferation and morphology. In co-culture with peripheral blood mononuclear cells (PBMCs), we found that MSCs not only lose their normal suppressive ability in high levels of palmitate, but actively support and enhance inflammation, resulting in elevated PBMC proliferation and pro-inflammatory cytokine release. The pro-inflammatory effect of MSCs in palmitate was partially reversed via palmitate removal and fully reversed through pre-licensing MSCs with interferon-gamma and tumor necrosis factor alpha. Thus, palmitate, a specific metabolic factor enriched within the T2D environment, is a potent modulator of MSC immunosuppressive function, which may in part explain the depressed potency observed in MSCs isolated from T2D patients. Importantly, we have also identified a robust and durable pre-licensing regimen that protects MSC immunosuppressive function in the setting of T2D.

AbstractsRodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1 week revealed better preservation of first‐phase glucose‐stimulated insulin secretion (GSIS) compared with cultured‐intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes (IL1B, CCL2, CXCL8). The expression of extracellular matrix markers (ASPN, COL1A1, COL4A1) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first‐phase GSIS. Lastly, the lentiviral‐mediated delivery of short hairpin RNA targeting glucokinase (GCK) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.

Mesenchymal stromal cells (MSCs) are administered locally to treat sites of inflammation. Local delivery is known to cause MSCs to aggregate into "spheroids," which alters gene expression and phenotype. While adherent MSCs are highly efficient in their inhibition of T cells, whether or not this property is altered upon MSC aggregation has not been thoroughly determined. In this study, we discovered that aggregation of MSCs into spheroids causes them to lose their T cell-suppressive abilities. Interestingly, adding budesonide, a topical glucocorticoid steroid, alongside spheroids partially restored MSC suppression of T cell proliferation. Through a series of inhibition and add-back studies, we determined budesonide acts synergistically with spheroid MSC-produced PGE2 to suppress T cell proliferation through the PGE2 receptors EP2 and EP4. These findings highlight critical differences between adherent and spheroid MSC interactions with human immune cells that have significant translational consequences. In addition, we uncovered a mechanism through which spheroid MSC suppression of T cells can be partly restored. By understanding the phenotypic changes that occur upon MSC aggregation and the impact of MSC drug interactions, improved immunosuppressive MSC therapies for localized delivery can be designed.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse β-cells, LDs are prominent in human β-cells. However, the regulation of LD mobilization (lipolysis) in human β-cells remains unclear. We found that glucose increases lipolysis in nondiabetic human islets but not in islets in patients with type 2 diabetes (T2D), indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets with shRNA targeting ATGL (shATGL) increased triglycerides (TGs) and the number and size of LDs, indicating that ATGL is the principal lipase in human β-cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS), and insulin secretion to 3-isobutyl-1-methylxanthine and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose-responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL-deficient INS1 cells and human pseudoislets showed reduced SNARE protein syntaxin 1a (STX1A), a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL-deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human β-cells and supports insulin secretion by stabilizing STX1A. The dysregulated lipolysis may contribute to LD accumulation and β-cell dysfunction in T2D islets.

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Tailoring MSCs to fit the disease. Fresh, cryopreserved and, prelicensed cryopreserved MSC are all being explored to treat numerous diseases, but all are not suitable to treat all conditions. injury. “*” denotes preferred therapeutic strategy when both fresh MSC and cryo‐MSC have shown utility in treating the disease but one is more efficacious or logistically suitable. Abbreviations: CLI, critical limb ischemia; GvHD. graft versus host disease; I/R, ischemia reperfusion (I/R); OI, osteogenesis imperfecta.