Dose and duration of interferon γ pre-licensing interact with donor characteristics to influence the expression and function of indoleamine-2,3-dioxygenase in mesenchymal stromal cells.

Human mesenchymal stromal cells (MSCs) are a leading cell therapy candidate for the treatment of immune and inflammatory diseases due to their potent regulation of immune cells. MSC expression of indoleamine-2,3-dioxygenase (IDO) upon interferon γ (IFNγ) exposure has been proposed as both a sentinel marker and key mediator of MSC immunomodulatory potency. Rather than wait for in vivo exposure to cytokines, MSCs can be pre-licensed during manufacturing to enhance IDO expression. In this study, we systematically examine the relative role that the dose of IFNγ, the duration of pre-licensing and the donor of origin play in dictating MSC production of functional IDO. We find that across three human MSC donors, MSCs increase their expression of IDO in response to both increased dose of IFNγ and duration of pre-licensing. However, with extended pre-licensing, the expression of IDO no longer predicts MSCs ability to suppress activated peripheral blood mononuclear cells. In addition, pre-licensing dose and duration are revealed to be minor modifiers of MSCs inherent potency, and thus cannot be manipulated to boost poor donors to the levels of high-performing donors. Thus, the dose and duration of pre-licensing should be tailored to optimize performance of specific donors and an emphasis on donor selection is needed to realize significant benefits of pre-licensing.

Aggregation of Human Mesenchymal Stromal Cells Eliminates Their Ability to Suppress Human T Cells.

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.

Nature vs. Nurture: Defining the Effects of Mesenchymal Stromal Cell Isolation and Culture Conditions on Resiliency to Palmitate Challenge.

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

IFN-γ and TNF-α Pre-licensing Protects Mesenchymal Stromal Cells from the Pro-inflammatory Effects of Palmitate.

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.