Therapeutic Efficacy of Interferon-Gamma and Hypoxia-Primed Mesenchymal Stromal Cells and Their Extracellular Vesicles: Underlying Mechanisms and Potentials in Clinical Translation

Multipotent mesenchymal stromal cells (MSCs) hold promises for cell therapy and tissue engineering due to their self-renewal and differentiation abilities, along with immunomodulatory properties and trophic factor secretion. Extracellular vesicles (EVs) from MSCs offer similar therapeutic effects. However, MSCs are heterogeneous and lead to variable outcomes. In vitro priming enhances MSC performance, improving immunomodulation, angiogenesis, proliferation, and tissue regeneration. Various stimuli, such as cytokines, growth factors, and oxygen tension, can prime MSCs. Two classical priming methods, interferon-gamma (IFN-γ) and hypoxia, enhance MSC immunomodulation, although standardized protocols are lacking. This review discusses priming protocols, highlighting the most commonly used concentrations and durations, along with mechanisms and in vivo therapeutics effects of primed MSCs and their EVs. The feasibility of up-scaling their production was also discussed. The review concluded that priming with IFN-γ or hypoxia (alone or in combination with other factors) boosted the immunomodulation capability of MSCs and their EVs, primarily via the JAK/STAT and PI3K/AKT and Leptin/JAK/STAT and TGF-β/Smad signalling pathways, respectively. Incorporating priming in MSC and EV production enables translation into cell-based or cell-free therapies for various disorders.


Introduction
Multipotent mesenchymal stromal cells (MSCs) are adult stem cells that can self-renew and differentiate into various mesenchymal cell lineages, including osteocytes, adipocytes, and chondrocytes [1].The high self-renewal capacity in vitro, multi-lineage differentiation potential, trophic factor secretion, and immunomodulatory properties of MSCs have made them popular biological candidates in cell therapies and tissue engineering in the past 30 years [2].Furthermore, the low expression of CD40, CD80, CD86, and major histocompatibility complex class I (MHC I), as well as the lack of MHC II expression, have rendered them immuno-privileged and therefore a highly valuable allogeneic cell therapy tool for regenerative medicine [3].MSCs were initially discovered in the bone marrow, but they have now been discovered in other tissues, including adipose tissue, muscle, peripheral blood, hair follicles, teeth, placenta, umbilical cord, and umbilical cord blood [1,[4][5][6].MSCs derived from different tissue sources displayed varying properties in terms of cellular composition (varying cell phenotypes, i.e., surface markers and immune profile), lineage-specific differentiation potential, and self-renewal capacities [7].In addition to their regenerative properties, MSCs exhibit robust systemic immunosuppressive effects via various mechanisms.Consequently, cell therapy based on MSCs is viewed as a promising approach for addressing autoimmune or inflammatory conditions, including graft-versus-host disease (GVHD), inflammatory bowel disease, multiple sclerosis, and coronavirus disease 2019 (COVID-19) [8].
Key mechanisms underlying MSC-based therapy primarily include three aspects.Firstly, cell replacement involves MSCs differentiating into various cell types to replace damaged tissues, integrating into affected areas.Secondly, immunomodulation occurs via MSCs' paracrine and extracellular vesicle secretion, regulating immune responses.Lastly, cell rescue involves MSCs transferring their organelles to injured cells via diverse mechanisms such as direct cell-to-cell contact and cell fusion [9].MSCs are known to exert their paracrine effects via a secretome comprising various soluble factors and extracellular vesicles (EVs), crucial for intercellular communication and signalling.Suppression of EV secretion resulted in MSCs losing their immunomodulatory effect [10].Furthermore, EVs' payload mirrors the molecular and functional properties of their producing cells [11].Although MSC therapies show promising therapeutic results in preclinical studies and clinical trials for a number of diseases, there are some challenges faced in the clinical applications of MSCs that have yet to be addressed.The inconsistency of MSCs in terms of immune compatibility, stability, heterogeneity, differentiation, and migratory capability are just a few of the factors that have contributed to the failure of MSC clinical development [12].The traits of MSCs are shaped by in vivo and in vitro biological, biochemical, and biophysical factors, which closely govern their functionality and survival.To date, numerous studies have shown that altering biological, biochemical, and biophysical factors can affect the fate of MSCs, their lineage-specific differentiation capability, and their functionality, eventually affecting their therapeutic potential in regenerative medicine [13][14][15].
According to Wang et al., MSCs are inert and incapable of dampening immune responses unless prompted by specific combinations of inflammatory cytokines [16].Cell priming, also known as preconditioning or licensing [7], is one of the commonly used strategies to augment MSC functionality and their EVs.Generally, MSCs can be primed with cytokines, hypoxia, growth factors, pharmacological or chemical agents, biomaterials, and different culture conditions [17].The characteristics of inflamed tissue, including low oxygen tension, elevated concentrations of inflammatory cytokines, and the presence of microorganisms, significantly influence the metabolism and functions of cells at the injury site.In these challenging environments, transplanted MSCs are expected to modulate the inflammatory and immune responses and facilitate the regenerative process.Consequently, there is a growing interest in preconditioning MSCs to enhance their physiology and fortify their therapeutic mechanisms.This approach aims to boost the number of cells reaching the site of inflammation, improve their survival, and enhance their anti-inflammatory and regenerative effects [18].
In the human body, oxygen availability in the tissues depends on the extent of vascularization and metabolic requirement of the tissue, often lower than the atmospheric oxygen levels (21%) [7].In the physiological state, the oxygen level in peripheral tissues, referred to as 'physioxia', typically ranges between 1% and 11% [23], and they physiologically adapt to this condition [7].In fact, MSC expansion in normoxic cell culture conditions (21%) may lead to cellular stresses that induce early senescence, DNA damage, and the extension of population doubling time [24][25][26].Studies have shown that hypoxia preconditioning at levels 0.5-5% can upregulate various bioactive factors such as interleukin 6 (IL-6), TNF-α, HGF, and vascular endothelial growth factor (VEGF), resulting in enhanced cell proliferation and tissue regeneration in animal models after transplantation [27][28][29].Moreover, hypoxia preconditioning has been observed to enhance the immunomodulatory capabilities of MSCs in various treatment scenarios [30][31][32][33].
Various priming methods, including inflammatory cytokines, hypoxia, 3D cultures, pharmacological or chemical agents, and biomaterials, have been discussed by Noronha et al. and Miceli et al. [7,34].In this review, inflammatory cytokine priming (IFN-γ) and hypoxia were chosen specifically due to their consistent effects in boosting MSC immunomodulatory function and growth factors secretion.This review presents recent findings from pre-clinical studies on priming MSCs and their EVs with IFN-γ and hypoxia.It highlights the most commonly used priming concentrations and durations and therapeutic effects (the relationship between in vitro or functional markers with therapeutic effects).The review also discusses underlying mechanisms and the feasibility of scaling up these methods to meet industrial requirements.
During the preparation of this work, the authors used ChatGPT 3.5 for grammar and spelling checks.After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the publication's content.

Phenotypic Characterization of Primed MSCs
Torkaman et al. observed no discernible alterations in the size, expansion, or morphology of the IFN-γ-primed human Wharton's Jelly MSCs (hWJ-MSCs), which exhibited characteristics resembling fibroblast-like cells with star-shaped and spindle morphology [52].Moreover, the presence of CD90, CD105, and CD73 markers was detected in both the primed and unprimed cells [43,52], while CD34, CD117, CD45, and CD31 markers were absent.This indicates that the isolated cells were mesenchymal stem cells, and the nature of hWJ-MSCs remained unchanged by IFN-γ, reaffirming that they retained their identity as mesenchymal stem cells [52].Human Leukocyte Antigen-DR isotype (HLA-DR), CD80, CD86, and CD40 are pivotal in the development of immune responses, as well as the initiation and persistence of graft rejection issues [52].Torkaman et al. showed that none of these surface molecules were expressed on primed and unprimed cells.However, Baudry et al. observed an increase in HLA-DR expression of the IFN-γ preconditioned human bone marrow-MSCs (BM-MSCs) [43,52].Both IFN-γ-primed and unprimed MSCs exhibited adipogenic and osteogenic differentiation capabilities, indicating that priming did not alter the cells' mesodermal characteristics [52,53].

IFN-γ Concentrations and Durations
Table 1 illustrates different IFN-γ priming concentrations and durations, influenced by factors such as MSC numbers, sources, culture protocols (e.g., mediums and serum supplements used and medium volume), and donor characteristics (age, health, and lifestyle).Bone marrow, umbilical cord and adipose tissues are the three main sources of MSCs in Table 1.However, no comparison was made between these sources.Nonetheless, all three MSC sources showed enhanced therapeutic effects upon the priming of different IFN-γ concentrations and durations.The use of U/mL as a concentration unit corresponds to IFN-γ specific activity but lacks a standardized method for determination.The concentrations of IFN-γ used are as low as 1 ng/mL and up to 50 ng/mL, whereas some studies used 500 U/mL and 200 IU/mL.It is noteworthy that the concentration units employed in these studies are not standardized, with some using ng/mL while others used U/mL or IU/mL.This inconsistency poses challenges for meaningful comparison between these study protocols.Rozier et al. demonstrated that extracellular vesicles (EVs) from MSCs pre-activated by a low dose of IFN-γ (1 ng/mL) were less efficient than naïve EVs, but when they increased the concentration to 20 ng/mL, improvement in several fibrotic, remodelling and inflammatory markers was observed [35].In another study, Atsma lab showed that both unstimulated MSCs and 500 U/mL IFN-γ-stimulated MSCs have no significant beneficial effect on cardiac function or remodelling in a mouse model of myocardial infarction (MI) [53].However, they previously reported that MSCs improved left ventricular function after acute myocardial infarction [54].These discrepancies highlight the need for further research to establish optimal priming conditions for MSCs with IFN-γ, considering variations in experimental protocols and conditions.
Furthermore, the priming durations differ between 8 h and 7 days whereby the most common are 24 and 48 h.In an Escherichia coli-induced pneumonia rat model, EVs derived from MSCs primed using 50 ng/mL IFN-γ for 8 h are sufficient to increase the survival rate and decrease lung injury severity [44].Nonetheless, Haan et al. showed no significant improvement even though the cells were primed using 500 U/mL IFN-γ for 7 days [53].Studies proved that priming with IFN-γ upregulates IDO expression of MSCs in a doseand time-dependent manner [41,55]; IDO is a key mediator of MSC immunomodulatory potency.However, prolonged exposure to IFN-γ can lead to MSCs' immunosuppressive potency reverting to levels similar to unprimed MSCs [55].
In conclusion, the use of IFN-γ for priming MSCs is an active area of research, and there is still some variability in the methods used across studies.Despite this variability, the general consensus seems to be that priming MSCs with IFN-γ concentrations ranging between 10 ng/mL and 100 ng/mL for a duration of 24 to 48 h can enhance the therapeutic potential of MSCs.However, further validation is needed to establish a standardized priming protocol that can be consistently applied across different studies and clinical applications.

The Therapeutic Effects of IFN-γ-Primed MSCs (The Relationship between In Vitro or Functional Markers with Therapeutic Effects)
To investigate the therapeutic potential of primed MSCs, the expression levels of various cell markers such as IDO, PGE 2 , IL-10, IL-6, HGF, VEGF, TGF-β, matrix metalloproteinase-3 (MMP-3), CXCL9, CXCL10, C-C motif chemokine ligand 8 (CCL8), and others were measured following IFN-γ and/or TNF-α priming.Numerous studies have reported that increased IDO expression following IFN-γ exposure can be a key indicator of MSC immunomodulatory potency [38][39][40][41]45,49,50,55], while Kim et al. also confirmed that the elevated IDO expression in MSCs stimulated by IFN-γ is a widespread occurrence that was observed in all MSCs tested, regardless of their source.The immunosuppressive properties of IDO involve tryptophan degradation, which is essential for T-cell proliferation [49].Takeshita et al. also found that in the presence of an IDO inhibitor, the inhibitory effects of IFN-γ-MSCs on PBMC proliferation were clearly diminished [45].In addition to IDO expression, activation of MSCs by IFN-γ can also be confirmed by the upregulation of MHC II and programmed death-ligand 1 (PD-L1) expression on MSCs [39].IFN-γprimed MSCs could secrete significantly higher levels of immunomodulation factors, i.e., IL-10, HGF, VEGF and TGF-β than unprimed MSCs [52].Additionally, the combination of IFN-γ and TNF-α stimulates MSCs to secrete a broad spectrum of pro-inflammatory and anti-inflammatory cytokines, including IL-4, IL-6, IL-10, and IL-13, promoting an anti-inflammatory phenotype [50].The upregulation of cytokines such as CXCL9, CXCL10, and CCL8 by IFN-γ-primed MSCs may also contribute to the recruitment of leukocytes and various immune responses [49].
Torkaman et al. reported that IFN-γ priming enhanced various immune suppression factors, including TGF-β, VEGF, HGF and IL-10, and significantly suppressed PBMC proliferation, leading to postponed clinical symptom onset in a mouse EAE model [52].This highlights the multiple roles of growth factors/cytokines.Various growth factors, such as HGF, VEGF, TGF-β, and HIF-1α, have been shown to be upregulated after IFN-γ priming [58][59][60][61].While VEGF and HGF are primarily associated with angiogenesis and proliferation, several studies have also indicated their involvement in the immunomodulatory properties of MSCs [19,52,[62][63][64].VEGF is believed to exert an immunosuppressive effect by inhibiting the migration or differentiation of bone marrow lymphoid precursors [64].On the other hand, HGF induces IL-10 expression in monocytes, suppresses Th1 and dendritic cell activities, and promotes the generation of IL-10-positive regulatory T cells.Additionally, HGF produced by MSCs facilitates the expansion of immune-suppressive myeloid-derived suppressor cells (MDSCs) [63].
Notably, IFN-γ and TNF-α priming rapidly activate PI3K/AKT signalling and drive glycolysis in human MSCs, significantly reducing inflammatory parameters in mice with inflammatory bowel disease.This suggests that glycolysis affects the expression of immunosuppressive effectors such as IDO and TNF-stimulated gene-6 (TSG-6), as well as regulates the anti-inflammatory function of MSCs [38].Conversely, Yao et al. found that IFN-γ priming shifts MSCs' energy metabolism towards aerobic oxidation, activating the JAK/STAT pathway and inducing IDO and PD-L1 production.Combined ATP and IFN-γ treatment showed enhanced therapeutic efficacy, with ATP boosting the immunosuppressive capabilities of IFN-γ-primed MSCs via JAK/STAT pathway activation [8].Metabolic programs profoundly affect immune cell responses; for instance, the rate of energetic consumption and biosynthesis of quiescent T cells is kept far lower than that of activated ones [65,66].Upon stimulation, CD8 + T cells will rapidly switch to a metabolic profile characterized by accelerated rates of glucose uptake, glycolysis, and biosynthesis [67].While the immunomodulatory functions of these immune cells are closely associated with distinct metabolic pathways, the metabolic requirements for the immunomodulatory function of MSCs remain to be fully clarified [38].
Despite their known enhanced immunomodulatory properties via paracrine effects, xenotransplantation of IFN-γ-primed human MSC extracellular matrix (ECM) complex (C-MSCγ) into an immunocompetent mouse calvarial bone defect model surprisingly induces bone regeneration, whereby lamellar bone was clearly observed in the lesion area [45].This suggests that IFN-γ-primed MSCs can attenuate undesirable xenogeneic immune responses, facilitating successful bone regeneration.Moreover, in a notable study by Zhang et al., it was shown that treating MSCs with IFN-γ and TNF-α effectively eliminated donor-dependent variations in immunomodulation.Transcriptomic analyses revealed a positive correlation between MSCs' immunomodulatory capabilities and the activation of IFN-γ and NF-κB signalling pathways induced by IFN-γ and TNF-α [22].Additionally, MSCs primed with both IFN-γ and kynurenic acid (KYNA) exhibit significant therapeutic efficacy in addressing acute colitis and chronic colon fibrosis in rats via the induction of IDO-1.IDO-1 facilitated cell homing, polarization of intestinal macrophages to the antiinflammatory M2 phenotype, and increased IL-10 expression to inhibit inflammation [40].
Other than that, numerous studies have harnessed the immunomodulatory property of MSCs for treating Type I diabetes, which is a well-known autoimmune disease characterized by specific adaptive immunity against β-cell antigens [50,68,69].For instance, Wang et al. demonstrated that cytokine-primed MSC-EVs exhibited high levels of the immune checkpoint molecule PD-L1, which significantly reduced CD4 + T cell density and activation via the PD-L1/PD-1 axis and promoted the transition of macrophages from the M1 to M2 phenotype in mice with Type I diabetes [69].Pancreatic islet transplantation is a therapeutic option for treating Type I diabetes; however, acute islet loss is a significant complication of this procedure.Barachini et al. reviewed various approaches using MSCs and MSC-EVs to create a more conducive immune microenvironment, aiming to reduce graft rejection and promote early vascularization to support graft survival [70].Mrahleh et al. reported that MSCs primed with IFN-γ and TNF-α exhibited an immunomodulatory effect on CD4 + and CD8 + T cells by producing tolerogenic dendritic cells, which inhibit antigen-specific T cell responses via induction T cell anergy [68].Similarly, Vaithilingam et al. reported improved encapsulated islet allograft survival and function via both coencapsulation and co-transplantation of islets with primed MSC [50].These findings have great implications for the future management and treatment of diabetes, which affects millions of patients worldwide [71].
In summary, while IFN-γ priming enhances therapeutic efficacy mainly via immunomodulatory properties and secretion of immunomodulatory factors, other reparative mechanisms also exist.Interestingly, studies show that IFN-γ and TNF-α priming induces glycolysis in MSCs, contributing to their anti-inflammatory properties [38].In contrast, other studies have reported that IFN-γ alone induces aerobic respiration in MSCs, which is linked to their immunosuppressive ability [8].Zhang et al. also demonstrated that treatment with IFN-γ and TNF-α can eradicate donor-dependent variations in MSC immunomodulation [22].Additionally, primed MSCs have been shown to induce bone regeneration, although the mechanism remains unclear.IDO is an immunosuppressive enzyme that enhances the catabolism of tryptophan to kynurenine.Both the depletion of tryptophan and the accumulation of toxic kynurenine inhibit T cell proliferation and reduce neuroinflammation in EAE mice [72].Therefore, the upregulation of MSC IDO expression and suppression of T-cell (PBMC) proliferation may serve as in vitro assays for assessing the immunomodulatory efficacy of IFN-γ priming in MSC and EV therapies.However, additional markers may also be evaluated to assess other therapeutic effects of interest that are concurrently influenced by IFN-γ priming.

Extracellular Vesicles Derived from IFN-γ-Primed MSCs
Comparative studies evaluating the influence of varying IFN-γ-preconditioning doses and durations on MSC-EV therapeutic efficacy are limited.One study compared the impact of different IFN-γ doses (1 ng/mL vs. 20 ng/mL) on EV therapeutic efficacy in systemic sclerosis in a murine model.They found that low-dose IFN-γ priming resulted in inferior suppression of dermal thickness and fibrosis by MSC-EVs compared to native EVs.However, primed EVs showed superior suppression of lung fibrosis and inflammation, partially ameliorating lung fibrosis.High-dose IFN-γ significantly enhanced EV-mediated amelioration of lung inflammation and fibrosis, with comparable effects on skin fibrosis suppression to native EVs [35].This underscores the importance of optimizing priming protocols for achieving optimal outcomes.Figure 1 illustrates the changes in EV cargoes following IFN-γ priming.
Overall, preconditioning MSCs with IFN-γ, alone or combined with other factors, affects EV release, cargo composition, immunomodulatory activity, and therapeutic potential.For example, IFN-γ priming, either alone or with TNF-α, increases EV secretion from menstrual blood-derived and adipose tissue-derived MSCs or human umbilical MSCs, respectively [21].Upregulated Rab27b and CD82 (involved in exosome secretion) may play a role in this observation [10].However, no major changes were marked in EV size, size distribution [11,57,73], the expression of common EV surface and intravesicular markers [11,39], and protein concentration [44] in response to IFN-γ priming.Conversely, other studies reported that IFN-γ priming of bone marrow MSCs decreased EV secretion [74] or increased CD9 and CD81 EV surface protein levels in primed EVs [47].Variances in the MSC tissue source, priming protocols, and MSC-EV separation techniques and storage conditions may account for these differences.Donor age may also influence MSC-EV secretion in response to priming.Cheng et al. demonstrated that pediatric MSCs produced more EVs and Rab27b than adult MSCs after IFN-γ + TNF-α stimulation [10].Profiling the proteomics of IFN-γ-primed MSC-EVs revealed a significant shift in EV expression of the immunomodulation-related proteins [10,11,39,48,81].Cheng et al. reported that 183 proteins were exclusive to the primed adipose tissue-MSC-EVs (AT-MSC-EVs) as compared to the resting AT-MSC-EVs, including TSG-6 and tumour necrosis factor-alpha-induced protein 3 (TNFAIP3) (i.e., A20), which are crucial mediators of the immunomodulatory activity of MSCs.These primed AT-MSC-EVs suppressed activated ConA-activated T cells (CD4 + ), but AT-MSC-EVs inhibitory activity was significantly lower than that of their primed origin cells and their secretome [10].Furthermore, Kim et al. identified a change in the proteomic cargo of IFN-γ-primed induced pluripotent stem cell-MSC (iPSC-MSC)-derived EVs, wherein 25 proteins were unique to the primed EVs, 101 protein expression significantly increased, and 181 protein expression significantly decreased.Additionally, primed EVs were enriched in proteins involved in immunoregulation pathways such as the JAK-STAT signalling pathway, T-cell and Treg-cell activation [81].
Serejo et al. found that IFN-γ preconditioning increased galectin-1, TGF-β, and IDO mRNA expression in adipose MSC-derived EVs but had no additional effect on EV immunoregulatory activity, as both IFN-primed and unprimed EVs inhibited T lymphocytes (in vitro) in a significant but comparable manner.Conversely, primed parent cells (adipose-MSCs) and their secretome showed a significantly greater T lymphocyte inhibition than Profiling the proteomics of IFN-γ-primed MSC-EVs revealed a significant shift in EV expression of the immunomodulation-related proteins [10,11,39,48,81].Cheng et al. reported that 183 proteins were exclusive to the primed adipose tissue-MSC-EVs (AT-MSC-EVs) as compared to the resting AT-MSC-EVs, including TSG-6 and tumour necrosis factor-alpha-induced protein 3 (TNFAIP3) (i.e., A20), which are crucial mediators of the immunomodulatory activity of MSCs.These primed AT-MSC-EVs suppressed activated ConA-activated T cells (CD4 + ), but AT-MSC-EVs inhibitory activity was significantly lower than that of their primed origin cells and their secretome [10].Furthermore, Kim et al. identified a change in the proteomic cargo of IFN-γ-primed induced pluripotent stem cell-MSC (iPSC-MSC)-derived EVs, wherein 25 proteins were unique to the primed EVs, 101 protein expression significantly increased, and 181 protein expression significantly decreased.Additionally, primed EVs were enriched in proteins involved in immunoregulation pathways such as the JAK-STAT signalling pathway, T-cell and Treg-cell activation [81].
Serejo et al. found that IFN-γ preconditioning increased galectin-1, TGF-β, and IDO mRNA expression in adipose MSC-derived EVs but had no additional effect on EV immunoregulatory activity, as both IFN-primed and unprimed EVs inhibited T lymphocytes (in vitro) in a significant but comparable manner.Conversely, primed parent cells (adipose-MSCs) and their secretome showed a significantly greater T lymphocyte inhibition than the unprimed MSCs [73], implying that IFN-γ priming may have a varying effect on MSC and their derived EV functionality.Similarly, Takeuchi et al. detected a higher expression of proteins involved in macrophage polarization (i.e., annexin-A1, lactotransferrin, galectin-3-binding protein, lactadherin, and aminopeptidase) by adipose MSC-EVs in response to IFN-γ priming.Accordingly, treating macrophages with IFN-γ-primed EVs raised the expression of anti-inflammatory macrophage factors (IL-10, Ym-1, Fizz-1, CD206) and decreased the expression of pro-inflammatory factors (IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1)) compared to the unprimed EVs, while both boosted macrophage motility and phagocytic activity in vitro [48].
Riazifar et al. showed that IFN-γ-MSC-EVs had a higher content of proteins with immunomodulating and neuroprotective characteristics, such as heat shock protein 70 (HSP70), macrophage inhibitory cytokine 1 (MIC-1), latent transforming growth factor -binding protein (LTBP) and galectin-1 (Gal-1).Profiling of RNA content revealed that, in comparison to resting MSC-EVs, IFN-γ-MSC-EVs contained an abundance of anti-inflammatory mRNAs such as IDO.Interestingly, both IFN-γ-MSC-EVs and unprimed MSC-EVs were found to be more enriched in non-coding RNAs (such as miRNA, lincRNA, and tRNA) than protein-coding RNAs relative to their parent cells, with the IFN-γ-MSC-EVs group showing significant enrichment of these noncoding RNAs compared with the unprimed MSC-EVs, implying their involvement in the regulation of several pathways.Interestingly, RNA functional inactivation by exposure to UV light led to a partial loss of MSC-EVs' function [39].
On the other hand, studies that profiled the miRNA expression of IFN-γ-primed MSC EVs yielded contradictory results.While some studies demonstrated that priming had a limited effect on miRNA expression [35,48,82], others demonstrated a major change in miRNA, suggesting a significant role of modulated miRNA in the functionality of IFNγ-primed EVs [21,39,47].Interestingly, Rozier et al. reported that IFN-γ downregulated the majority of the modified miRNAs in MSCs.In contrast, no miRNA was found to be downregulated in IFN-γ-primed EVs.This study also revealed that IFN-γ -preconditioning differently altered the mRNA levels of several anti-inflammatory factors in MSCs and their generated EVs [35].
Furthermore, several studies demonstrated enhanced therapeutic potency of MSC-EVs with IFN-γ-priming in animal models of other conditions, including colitis [47], acute lung bacterial-induced pneumonia [44], cirrhosis [48], atopic dermatitis [81], and MI [21], highlighting the potential of IFN-γ-priming in enhancing MSC-EV therapeutic potency.For instance, treating the colitis mice model with IFN-γ-MSC-EVs considerably modified the condition indices and histological grade, along with reduced Th17:Treg cell ratios.In vitro, MSC-EVs inhibited Th17 differentiation, with IFN-γ-MSCs-EVs exhibiting a better inhibitory activity.This effect was suggested to be due to the upregulation of miR-125a and miR-125b in IFN-γ-primed EVs, which negatively regulated Stat3 and p-Stat3, leading to Th17 differentiation suppression [47].
To summarize, limited studies have explored the impact of IFN-γ pre-activation of MSCs on EV cargo and therapeutic efficacy, indicating a need for further research in this area.Evidence suggests that IFN-γ priming, alone or combined with other factors, enhances the expression of immunoregulatory factors in released EVs, potentially enhancing the therapeutic potential of MSC-EVs for immunological diseases.However, the effects of IFN-γ priming on EVs may vary depending on factors such as MSC tissue source, donor age, and priming protocol.While in vitro immunomodulatory effects of primed IFNγ-EVs were inconsistent, better and more consistent outcomes were observed in vivo.Further research is necessary to understand the mechanisms underlying EV therapeutic functionality.Additionally, more studies are needed to compare the efficacy of primed MSCs and their derived EVs, as previous studies yielded conflicting results.Cargo analysis suggests potential distinct mechanisms of action between MSCs and EVs post-IFN-γ priming.Furthermore, the existing data suggest that IFN-γ-primed EVs act primarily on modulating immune cells such as macrophages [44,48] and T lymphocytes [39,47], an observation that warrants further investigation.

Long-Term Safety and Efficacy of IFN-γ Priming
In an equine model of osteoarthritis, Barrachina et al. conducted a 6-month monitoring study and noted that the beneficial effects of primed MSCs appeared to be short-lived.This may be attributed to the limited lifespan of MSCs following in vivo administration, particularly in cases of allogeneic and MHC-mismatched cells.Initially, both naïve and primed MSC treatments demonstrated an anti-inflammatory effect shortly after administration, especially after the initial injection when joint inflammation was more prominent.However, upon the second injection of allogeneic MSCs, a slight transient inflammatory reaction occurred, suggesting heightened immunogenicity of these cells.Notably, this reaction was observed only after the second injection, potentially indicating the development of immune memory, as evidenced by the recent discovery of functional antibodies against MHC-mismatched MSCs in horses [36].In a GVHD mouse model, while all mice that were transplanted with hPBMCs alone or with a combination of hPBMCs and MSCs once had died, approximately 20% of mice that were transplanted twice with MSCs survived at 8 weeks post-transplantation. Notably, there seemed to be a trend suggesting a survival advantage among mice co-transplanted with hPBMCs and IFN-γ-primed MSCs compared to those receiving hPBMCs and naïve MSCs together.In this group, survival rates ranged between 40% and 60% at the 8-week post-transplantation mark [49].Furthermore, in a study by Vaithilingam et al. in a diabetic mouse model, it was observed that all mice that received primed MSCs remained normoglycemic, in contrast to 71.4% of those in the unprimed MSC group at day 50 post-transplantation.Moreover, the viability of islets co-encapsulated with primed MSCs was significantly higher compared to those with unprimed MSCs at the same time point [50].Additionally, Torkaman et al. demonstrated that neurologic functional recovery in EAE mice was significantly improved 50 days post-immunization with IFN-γ-primed MSCs compared to unprimed MSCs [52].Safety monitoring in these studies was based on the premise that there were no manifestations of adverse health conditions.However, long-term systemic toxicity studies, including histopathological evaluation of vital organs and blood biochemical analysis, are lacking.

Signalling Pathways and Mechanisms of Action of IFN-γ Priming
Understanding the underlying mechanism of MSC therapy via the various signalling pathways is critical as it paves the way for a strong foundation for future scientific research and clinical applications for a variety of diseases.Moreover, each priming method exhibited distinct enrichment of molecular and cellular functions, which may explain their diverse mechanisms of action [37].A schematic diagram of these pathways is illustrated in Figure 2.
Kim et al. demonstrated that IFN-γ induces IDO expression in MSCs via the Janus kinase/signal transducer and activator of transcription 1 (JAK/STAT1) signalling pathway, where both JAK1/2 and STAT1 in MSCs were activated following IFN-γ-stimulation [49].IDO plays a direct role in the immunosuppressive properties of MSCs by suppressing antigen-driven T-cell proliferation.JAKs are associated with cytokine receptors, which are activated upon stimulation, and they phosphorylate STAT proteins, enabling them to be transported to the nucleus and thus regulate the transcription of target genes [83].Nonetheless, activation of Toll-like receptor 3 (TLR3) in MSCs rarely induces IFN-β and/or IDO expression, suggesting that TLR signalling is not a major pathway in MSC immunosuppressive functions [49].Moreover, Yao et al. also showed that IFN-γ-induced metabolic reconfiguration to aerobic oxidation activates the JAK/STAT pathway, which is necessary for expressing IDO and PD-L1 in MSCs [8].Similarly, Ling et al. also reported that IFN-γ-MSCs could reduce inflammation in experimental autoimmune encephalomyelitis (EAE) mice via the forkhead box P3 (Foxp3)/retinoic acid-related orphan receptor gamma t (ROR-γt)/STAT3 signalling pathway, whereby this is the downstream pathway of JAK/STAT signalling [42].This pathway involves Foxp3 directly interacting with ROR-γt and STAT3 signalling, crucial for inducing ROR-γt and subsequent T helper 17 (Th17) cell differentiation [84].The imbalance of Th17/Tregs is implicated in the pathogenesis of EAE [42].
Analysis of global mRNA profiles from MSCs primed with poly I:C or IFN-γ revealed enrichment of canonical pathways associated with cell survival and inflammatory responses, including interferon signalling and the antigen-presenting pathway, compared to non-primed MSCs.At the same time, IFN-γ priming has been shown to affect much more complex functions, including cellular development, cell growth and proliferation, cell-to-cell signalling and interactions, cell-mediated immune responses and cell movement [37].Moreover, Riazifar et al. used an ingenuity pathway analysis database of gene ontology and demonstrated that the phosphoinositide 3-kinase/Ak strain transforming (PI3K/AKT) signalling pathway is the most relevant canonical pathway affected by Signalling pathways underlying MSCs priming with IFN-γ or hypoxia.IFN-γ priming activates the JAK/STAT and PI3K/AKT pathways, eventually enhancing the immunomodulation properties of MSCs upon priming.In contrast, hypoxia priming activates the Leptin/JAK/STAT pathway and inhibits the TGF-β/Smad pathway, thus empowering the angiogenesis and anti-fibrotic properties of MSCs after priming.IFN-γ, interferon-γ; JAK, Janus kinase; STAT, signal transducer and activator of transcription; P, phosphate group; PI3K, phosphoinositide 3-kinase; Akt, Ak strain transforming; mTOR, mammalian target of rapamycin; Foxp3, forkhead box P3; ROR-γt, retinoic acidrelated orphan receptor gamma t; IDO, indoleamine 2,3-dioxygenase; TSG-6, tumour necrosis factor (TNF)-stimulated gene-6; TGF-β, transforming growth factor-beta; Smad, suppressor of mothers against decapentaplegic; HIF-1α, hypoxia-inducing factor-alpha; VEGF, vascular endothelial growth factor.Created with BioRender.com(accessed on 17 May 2024).
Analysis of global mRNA profiles from MSCs primed with poly I:C or IFN-γ revealed enrichment of canonical pathways associated with cell survival and inflammatory responses, including interferon signalling and the antigen-presenting pathway, compared to non-primed MSCs.At the same time, IFN-γ priming has been shown to affect much more complex functions, including cellular development, cell growth and proliferation, cellto-cell signalling and interactions, cell-mediated immune responses and cell movement [37].Moreover, Riazifar et al. used an ingenuity pathway analysis database of gene ontology and demonstrated that the phosphoinositide 3-kinase/Ak strain transforming (PI3K/AKT) signalling pathway is the most relevant canonical pathway affected by miRNAs enriched in IFN-γ-primed exosomes, as 19 out of 123 genes in this pathway are affected [39].Similarly, Xu et al. also showed that TNF-α and IFN-γ acutely activate the PI3K/AKT signalling pathway, essential for the expression of IDO and TSG-6.TSG-6, secreted from stimulated MSCs, binds to hyaluronan and reduces inflammation in various disease models [38].The activation of the PI3K/Akt pathway begins with IFN-γ binds to its receptor and then activates JAK2 to phosphorylate STAT1, where phosphorylated STAT1 further activates PI3K and continues with the recruitment and activation of the inactive signalling protein AKT [75].
Numerous studies have indicated that IFN-γ-primed MSCs suppress T cell proliferation, regulate the activity of helper T cells (Th), and induce regulatory T cells (Tregs), suggesting that T cells play a critical role in the immunomodulatory properties of primed MSCs [37,39,42,45,[47][48][49]51,52].Yang et al. showed that increased levels of miR-125a and miR-125b in primed MSC-EVs inhibit Th17 cell differentiation, leading to increased efficacy in treating colitis [47].Additionally, the upregulation of anti-inflammatory cytokines IL-4, IL-6, IL-10 and granulocyte colony-stimulating factor, as well as enhanced nitric oxide production, help modulate the immune response and improve the immunosuppressive capacity of MSCs [50].Baudry et al. reported that increased red blood cell velocity, rolling white blood cell flux and number of venules with circulating white blood cells, as well as reduced rate of soluble E-selectin (which may serve as a biomarker to monitor an organ's endothelium damage), can improve microvascular hemodynamics in early stages of sepsis [43].
In an EAE mouse model, the IFN-γ-primed exosomes were not detected after a short period in vivo, suggesting a "hit and run" mechanism for their long-lasting efficacy, likely via immunomodulatory mechanisms such as induction of Tregs for immune tolerance [39].Furthermore, Park et al. showed that IFN-γ-primed MSCs regulate Aspergillus fumigatusinduced immune responses via the regulation of Th17 immune responses, whereas poly I:C-primed MSCs control both eosinophil-associated Th2 immunity and neutrophil-related Th17 immunity [37].Other than that, Varkouhi et al. reported that the beneficial effects of MSC-EVs appear to be driven by the enhancement of macrophage phagocytosis and killing of Escherichia coli [44].In an inflammatory bowel disease mouse model, primed MSCs reduced inflammatory parameters via enhanced IDO and TSG-6 expression, promoting glycolysis, glucose uptake, and hexokinase II activity [38].Additionally, Ye et al. showed that IFN-γ and KYNA combination induces IDO-1 expression, facilitating cell homing, M2 polarization of intestinal macrophages, and increased IL-10 expression, effectively inhibiting inflammation in acute colitis and chronic colon fibrosis [40].
In summary, research on IFN-γ priming of MSCs elucidated its mechanisms and therapeutic implications across various diseases.IFN-γ triggers IDO expression in MSCs via the JAK/STAT1 pathway, facilitating immunosuppression and T-cell regulation.Moreover, the PI3K/AKT pathway plays a crucial role in mediating IDO and TSG-6 expression.IFN-γ-primed MSCs exhibit diverse effects, impacting cellular functions and canonical pathways associated with survival and inflammation.Notably, IFN-γ priming influences T cell activities, promotes Treg induction, and modulates cytokine production.Furthermore, the "hit and run" mechanism of IFN-γ-primed extracellular vesicles underscores their long-lasting immunomodulatory effects.

MSCs Priming with Hypoxia
Several studies have demonstrated the effects of conditioning MSCs in hypoxic environments, contrasting with standard cell culture conditions of 20-21% oxygen levels.Table 2 outlines key findings from preclinical studies focusing on priming MSCs in reduced oxygen environments.This method aims to enhance MSC proliferation and longevity while also amplifying their angiogenic and immunomodulatory capacities.

Phenotypic Characterization of Primed MSCs
Wu et al. found that long-term hypoxic treatment did not alter the stem cell characteristics of human UC-MSCs, including morphology, surface biomarker expressions, differentiation ability, tumorigenicity, and chromosomal stability [30].Similarly, Kim et al. demonstrated that the expression of MSC surface marker proteins (CD73, CD90, CD105, and CD166) and multipotency remained comparable between small MSCs primed with hypoxia and calcium ions (SHC-MSCs) and naïve MSCs [33], suggesting that hypoxia preconditioning did not induce significant alterations in MSC characteristics.However, in contrast, Hu et al. reported that mouse BM-MSCs exposed to 5% oxygen exhibited a notably more flattened spindle-shaped morphology, less convex compared to those in 21% oxygen [88].

Hypoxia Priming Methods (Oxygen Levels) and Durations
Oxygen levels in these studies ranged from 0.5% to 5%, with 1% and 5% being the most common.Additionally, 1% oxygen tension has been frequently used in acute kidney injury (AKI) and renal ischemia reperfusion injury (IRI) disease models [76][77][78], whereas 5% oxygen tension has been frequently used in diseases related to immunomodulation which included allotransplantation, traumatic brain injury (TBI), skin-wound healing and chronic asthma [32,79,80,87]; yet there is no direct relationship observed between oxygen tension and disease models.Moreover, the durations of priming in these studies typically range from 24 h to 72 h.However, Kim et al. kept the MSCs under hypoxic conditions (3% O 2 ) in media supplemented with calcium ions throughout the entire culturing process [33], similar to Wu et al., who cultured the MSCs under 1% O 2 throughout the entire culturing process [30].Additionally, the three primary sources of MSCs listed in Table 2 are the bone marrow, umbilical cord, and adipose tissues.While no direct comparison was conducted, all three sources exhibited improved therapeutic effects when subjected to priming with different oxygen levels and durations.
Interestingly, Kim et al. utilized small MSCs primed with hypoxia (3% O 2 ) and 1.8 mM calcium ions (SHC-MSCs), isolated by filtering MSCs through a pluriStrainer with a pore size of 10 µm.Small MSCs were selected because they are readily self-renew whereas large cells tend to lose this characteristic, and they are less likely to become trapped in capillaries, which would be beneficial for its clinical application.They reported that SHC-MSCs have an enhanced potency for treating GVHD as compared to naïve MSCs [33].Moreover, Ishiuchi et al. demonstrated that serum-free medium and hypoxia preconditioning (1% O 2 ) synergistically enhanced the proliferative capacity and anti-fibrotic effects of MSCs [77].These results suggested that serum-free conditions and hypoxic preconditioning do not antagonize each other but enhance paracrine activity.Hypoxia preconditioning improved the direct anti-fibrotic effect of serum-free MSCs but did not enhance their anti-inflammatory effect, likely due to pre-existing strong anti-inflammatory effects of serum-free MSCs.Surprisingly, Soares et al. compared priming MSCs with hypoxic condition (5% O 2 ) or 1000 units of IFN-γ for 72 h and found that hypoxic priming is significantly better than IFN-γ priming in prolonging allograft rejection [79].One of the possible explanations is that IFN-γ priming will convert MSCs to an antigen-presenting cell phenotype, thereby decreasing their immunomodulatory capability [89].
In summary, the most commonly used hypoxic conditions and durations were 1% oxygen level and 24 h, respectively.Yet there is no standard protocol for this hypoxia preconditioning, and hence, more studies can be carried out to compare the effect of different hypoxic conditions and durations on enhancing MSC therapies.

Therapeutic Effects of Hypoxia-Primed MSCs (The Relationship between In Vitro or Functional Markers with Therapeutic Effects)
To investigate the therapeutic efficacy of MSCs after hypoxia priming, the expression level of a few cell markers was measured, which include hypoxia-inducible factor (HIF)-1, leptin, basic fibroblast growth factor (bFGF), VEGF, IDO, HGF, PGE 2 and more.HIF-1, a dimeric protein complex crucial for the body's response to low oxygen levels, plays a key role in vascularization in hypoxic areas like localized ischemia and tumours [90].It serves as the upstream regulator of several key genes, including VEGF, HGF, PGE 2 , and IDO [91].Studies have consistently shown the induction of hypoxia-inducible factor-alpha (HIF-1α) expression in MSC cultures under hypoxic conditions [92,93].Additionally, hypoxic MSCs exhibit enhanced levels of angiogenic factors such as VEGF and bFGF, along with improved antioxidative capacity.Paracrine effects seem to be the main mechanism by which hypoxic MSCs modify kidney effects of ischemia-reperfusion injury (IRI) [78].Similarly, other studies also showed that hypoxia-preconditioned MSCs enhance the production of bFGF, VEGF, HGF and PGE 2 [76,77,87].Knockdown experiments suggest that VEGF acts as an upstream effector of HGF, and both VEGF and HGF knockdown diminish the anti-fibrotic effect of hypoxia-preconditioned MSCs [76,77].
Numerous studies have conducted various functional tests to further establish the effectiveness of hypoxia priming.These include MSC migration assays [77,79,85], MSC apoptosis assays [85], tube formation assays [33,85], assessments of human T-cell (PBMC) proliferation [33,79], macrophage polarization assays [77], measurements of oxygen consumption rate (OCR), mitochondrial membrane potential, reactive oxygen species (ROS) levels [86] and more.Hypoxia preconditioning induces CXCR4 expression in a leptindependent manner, enhancing MSC homing, survival, and angiogenesis, ultimately improving cardiac function in a mouse model of myocardial infarction (MI) [85].Intriguingly, hypoxia priming upregulates IDO expression, inhibiting the proliferation of CD4 + T cells and boosting the motility and proliferative potential of MSCs.This, in turn, delays the onset of acute rejection while preserving the recipient's adaptive immune response [79].Surprisingly, hypoxia priming also enhances the expression of Parkinson's disease protein 7 (PARK7/DJ-1) in its EVs, resulting in the alleviation of mitochondrial damage and the suppression of angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) degradation [86].Prolonged hypoxia exposure improves MSC proliferative capacity and telomerase activities, reduces senescence, and preserves multipotency compared to normoxic conditions.Hypoxic EVs inhibit immune activation via VEGF-mediated inhibition of dendritic cell maturation and downregulation of costimulatory molecules and HLA-DR expressions [30].
Interestingly, priming MSCs in hypoxic environments (5% O 2 ) before IRI resulted in a 4.3-fold increase in IDO transcript expression compared with normoxic MSCs (21% O 2 ).The addition of a competitive IDO inhibitor to the hypoxia-primed MSCs, unexpectedly, did not significantly affect allograft survival, while hypoxia-primed MSCs are better than IFN-γ priming at preventing allograft rejection.This suggests that hypoxic conditions may drive IDO expression to a level where the competitive inhibitor no longer effectively saturates enzymatic binding sites [79].Remarkably, Hendrawan et al. demonstrated that conditioned medium (CM) from hypoxic MSCs facilitated wound repair more effectively in early-stage diabetic wound models compared to antibiotic treatment commonly used for diabetic foot ulcer care [87].However, in an atherosclerotic renal artery stenosis (ARAS) porcine model, Farooqui et al. reported that hypoxia preconditioning of MSCs showed comparable effects to normoxia and did not enhance the therapeutic impact of MSCs on renal function, despite inducing a reduction in DNA hydroxymethylation (5hmC levels) of inflammatory and profibrotic genes [31].
Additionally, Nowak-Ste ¸pniowska et al. found that 19 out of 30 conditions (varying in oxygen concentration and incubation time) led to increased MSC proliferation in oxygen concentrations ranging from 1 to 5%.The higher proliferation in hypoxic conditions may result from the shift to anaerobic respiration, leading to increased glucose consumption and lactate generation in MSCs [94].Hypoxia also diminishes cellular ATP consumption and the production of ROS, hence preventing bioenergetic collapse [95].Intriguingly, Xu et al. reported a non-invasive approach involving nebulized hypoxic EV inhalation, significantly reducing chronic airway inflammation and remodelling.The EVs predominantly accumulate in the lungs and persist for a duration of 7 days [32].
In conclusion, HIF-1 expression serves as an indicator of the effectiveness of hypoxia priming in MSC therapies.Hypoxia priming enhances MSC properties including angiogenesis, anti-fibrotic effects, anti-apoptosis, and cell homing while also suppressing inflammatory cell infiltration via IDO expression and improving MSC antioxidative capacity.Additionally, hypoxia-primed MSCs exhibit a shift from aerobic to anaerobic respiration and increased glucose uptake.

Extracellular Vesicles Derived from Hypoxia-Primed MSCs
There is a scarcity of comparative studies assessing the impact of varying oxygen levels and durations on the therapeutic efficacy of MSC-EVs.In general, hypoxic EVs have demonstrated better therapeutic efficacy compared to normoxic EVs [30,86].Wu et al. reported that both hypoxic EVs and conditioned medium (CM) exhibit more pronounced therapeutic effects than their normoxic counterparts, suggesting that the essential components in the CM likely originate from EVs [30].Studies have indicated that there is no significant difference in average size and marker protein expression between normoxic EVs and hypoxic EVs [30,86,96], revealing that hypoxia priming does not induce significant alterations in the characteristics of MSC-EVs.Interestingly, Xu et al. designed an inhalation device for asthma mice, showing that nebulized hypoxic EVs and hypoxic EVs have similar round nanoparticle structures and complete membranous integrity, indicating maintained structural integrity of nebulized hypoxic EVs [32].
There are indications that the induction of HIFs could directly impact the pathways of EV biogenesis involving Rabs [97].In tumour B cells, HIFs were shown to directly bind to the Rab22A locus, leading to the expression of Rab22A, a protein necessary for the budding of microvesicles from the plasma membrane.Silencing Rab22A expression prevented hypoxia-induced EV release [98].Andrew et al. demonstrated that hypoxic priming increased EV release, while inflammatory priming (TNF-α and IFN-γ) affected EV size [99].Similarly, in a review paper, Bister et al. reported that quantitative measurements of EVs provided evidence of increased EV release after hypoxia compared to normoxia.However, the exact mechanisms controlling the fate of multivesicular bodies (MVBs) or the release of EVs in response to hypoxia in other cellular contexts still need clarification [97].
In an osteoarthritis rat model, Zhang et al. reported that miRNA sequencing indicated changes in miRNA expression due to hypoxia pretreatment.Specifically, 12 miRNAs, including miR-181c-5p, miR-18a-3p, miR-376a-5p, and miR-337-5p, showed significant alterations.Hypoxia priming was demonstrated to decrease the expression of these miRNAs, and their target genes, such as ALB, STAT3, and MAPK1, are associated with osteoarthritis and cartilage repair [96].Furthermore, Zhuang et al. illustrated that the expression of miR-210-3p, miR-322-5p, miR-3574, miR-503-5p, miR-322-3p, and miR-450a-5p significantly increased in hypoxic small EVs compared to normoxic small EVs, while the expression of miR-22-5p and miR-499-5p was inhibited.Importantly, previous evidence has suggested that miR-210 may play a role in the angiogenic process in various cell types, and it is known to be hypoxia-responsive, consistent with the sequencing results [100].
Furthermore, Lu et al. conducted a quantitative proteomics analysis to discern differential protein expressions between normoxic EVs and hypoxic EVs.They identified PARK7/DJ-1 as the protein with the most significant expression difference, with higher levels in hypoxic EVs.DJ-1 plays a role in modulating anti-oxidative stress and cardiac hypertrophy [86].Additionally, Xu et al. observed that hypoxic EVs exhibited greater protein diversity compared to normoxic EVs, identifying a total of 395 proteins, including 74 unique ones.Pathway analysis of these unique proteins revealed enrichment in pathways related to angiogenesis, such as "cell migration" and "cell adhesion", along with several inflammatory pathways.Intriguingly, they observed reduced EV secretion levels when cells were exposed to both nutritional starvation and oxygen-depleted conditions [101].The changes in EV cargoes following hypoxia priming were also illustrated in Figure 1.
In summary, limited studies have delved into the impact of diverse oxygen levels and durations on MSCs concerning EV cargo and therapeutic effectiveness, indicating a necessity for further investigation in this realm.The existing evidence suggests that hypoxia priming, involving variations in oxygen levels and durations, enhances the expression of therapeutic factors in released EVs, presenting a potential avenue for amplifying the therapeutic efficacy of MSC-EVs.Notably, the influence of hypoxia priming on EVs may exhibit variations contingent on factors such as MSC tissue source, donor age, and the priming protocol employed.Additional studies are imperative for a comprehensive understanding of the mechanisms underpinning the therapeutic functionality of EVs.Furthermore, cargo analysis of both MSCs and EVs supports the likelihood of distinct mechanisms of action, as the impact of hypoxia priming on the cargo of MSCs and EVs may differ.

Long-Term Safety and Efficacy of Hypoxia Priming
In a humanized mouse model of GVHD, the administration of SHC-MSCs led to improved survival, reduced weight loss, and decreased histological evidence of GVHD 6 weeks post-transplantation. Eight weeks after transplantation, approximately 90% of mice transplanted with human PBMNCs alone (GVHD group) had died, whereas the majority of mice transplanted with naïve MSCs (80%), SHC-MSCs (90%), and Polo-like kinase-1 (PLK1)-overexpressing MSCs (90%) survived.In line with the anti-inflammatory and immunomodulatory activities observed in vitro, the levels of human inflammatory cytokines, including IL-2, TNF-α, and IFN-γ, were more effectively reduced in the blood samples of GVHD mice treated with SHC-MSCs compared to those treated with naïve MSCs [33].Long-term safety has not been established in the study.

Signalling Pathways and Mechanisms of Action of Hypoxia Priming
In an MI mouse model, Hu et al. showed that leptin signalling is a critical initial step in the enhanced survival, chemotaxis, and therapeutic capabilities of MSCs imparted by hypoxia preculture [85].HIF-1α is a molecular sensor of hypoxia, and it transactivates the leptin gene expression by HIF-1α transcription-dependent activity.Leptin is a product of a hypoxia-inducible gene, and hypoxia differentiates the preadipocytes into leptin-secreting endocrine cells in an mTOR-dependent manner [102].Leptin, a multifunctional cytokine primarily produced by adipocytes, regulates various processes, including appetite control, energy balance, metabolism, cell survival, migration, and angiogenesis [103][104][105].Upon binding to its transmembrane receptors (ObR), leptin triggers the autophosphorylation of two JAK molecules, leading to the activation of STAT proteins via tyrosine phosphorylation [106].Hence, leptin can also activate STAT-independent pathways, including extracellular signal-regulated kinase (ERK) and PI3K cascades [107].Moreover, the possible mechanisms involved in this leptin signalling are STAT3/HIF-1α/VEGF signalling and stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signalling that leads to enhanced MSC engraftment, and cardiac protection via autocrine and paracrine effects as well as possibly recruitment of endogenous progenitor cells.Their findings support the hypothesis that STAT3 is part of the leptin signal pathway and plays an important role in enhanced cardio-protection by hypoxia priming.STAT3 enhances the HIF-1α actions by binding and stabilizing the transcription factor complex, and this is required for sufficient activation of several hypoxia-activated genes, including VEGF [108], while SDF-1/CXCR4 signalling promotes cell homing and survival where the induction of CXCR4 expression is dependent, at least in part, on HIF-1α [109].
Other than that, Ishiuchi et al. found that MSC-conditioned medium inhibits TGFβ/Suppressor of Mothers Against Decapentaplegic (Smad) signalling as a mechanism by which MSCs exert their anti-fibrotic effect.The conditioned medium from 1% O 2 (hypoxia) MSCs contained HIF-1α that further suppressed TGF-β1-induced phosphorylation of Smad2 and alpha-smooth muscle actin (α-SMA) compared with 21% O 2 (normoxic) MSCs [76].Numerous studies demonstrated that TGF-β is the key mediator in chronic kidney diseases associated with progressive renal fibrosis, where TGF-β1 will activate Smad2 and Smad3 that bind directly to several microRNAs to either negatively or positively regulate their expression and function in renal fibrosis [110].Similarly, subsequent studies found that hypoxia and serum-free medium-cultured MSCs (hypo-SF-MSCs) also inhibit TGF-β/Smad signalling, with HGF knockdown attenuating this effect [77].A schematic diagram of these pathways is illustrated in Figure 2.
Fascinatingly, Lu et al. documented that PARK7/DJ-1 (Parkinson's disease protein 7) derived from hypoxic EVs mitigates mitochondrial damage and inhibits proteasome subunit beta type 10 (PSMB10) activity via direct interaction.This reduces AT1R-associated protein (ATRAP) degradation, thus inhibiting AT1R-mediated signalling and mitigating cardiac hypertrophy [86].DJ-1 is vital for maintaining mitochondrial function and acting as an endogenous antioxidant, offering cardiovascular protection [111,112].In a mouse model of allergic rhinitis (AR), extended exposure to hypoxia has been observed to elevate VEGF levels in EVs.This increase, in turn, suppresses the differentiation and maturation of dendritic cells, which are pivotal in the pathogenesis of AR [30].Intriguingly, Soares et al. illustrated that hypoxia priming, similar to IFN-γ priming, enhances IDO expression, protecting endothelial cells, inhibiting CD4 + T cell proliferation, and boosting motility and proliferation potential [79].
In conclusion, leptin signalling plays a crucial role in enhancing the survival, chemotaxis, and therapeutic capabilities of MSCs induced by hypoxia preconditioning.Leptin, primarily produced by adipocytes, activates various pathways, including JAK/STAT, ERK, and PI3K cascades upon binding to its receptors.Moreover, an MSC-conditioned medium has been shown to inhibit TGF-β/Smad signalling, further suppressed under hypoxic conditions, offering anti-fibrotic effects.PARK7/DJ-1 from hypoxic EVs mitigates mitochondrial damage and inhibits PSMB10 activity, reducing ATRAP degradation, thereby inhibiting AT1R-mediated signalling and mitigating cardiac hypertrophy.

Primed MSC in Clinical Trials
Establishing standardized and consistent markers for evaluating the efficacy of IFN-γ and hypoxia priming is essential for ensuring reproducible and reliable results across different studies.Key potency markers such as IDO, PGE2, VEGF, TGF-β, and HIF-1 should be incorporated into the product validation protocol, along with functional assays like activated T cell inhibition before clinical use.Consistency in marker selection is vital for meaningful comparison of results between studies, facilitating a clearer understanding of the priming mechanisms and optimal conditions for MSC-based therapies.It is also crucial to note that while some studies have shown positive results with IFN-γ and hypoxia priming, others have reported limited or no improvement when compared to unprimed MSCs [31,35,46,51,53].This highlights the importance of further research to fully understand the multiple mechanisms at play and determine the conditions under which IFN-γ and hypoxia priming provide the best therapeutic outcomes.In addition, IFN-γ priming can be combined with other priming strategies, such as hypoxia.For example, an in vitro study reported that hypoxia is a relatively low-cost addition to IFN-γ priming that leads to additive immunosuppressive effects over IFN-γ priming alone [113].
According to the ClinicalTrials.govdatabase, there are two ongoing phase 1 clinical trials using IFN-γ-primed MSCs for xerostomia following radiotherapy and asthma started in the year 2022.Both trials employ IFN-γ-primed MSCs derived from human bone marrow, although the specific method and duration of IFN-γ priming have not been revealed.The investigators claimed that the risk of cell therapy using IFN-γ-primed MSCs is comparable to that of naïve MSCs, which possess an outstanding and well-established safety profile [114].The Good Manufacturing Practice (GMP) protocol for IFN-γ-primed MSCs has been developed and demonstrated to be safe, feasible, and reliable in preclinical models.The upscaling of the production of IFN-γ-primed MSCs has also been shown to be feasible in this GMP setting.The two-step approach involves MSC isolation, expansion and cryopreservation, followed by thawing and priming with IFN-γ for 7 days before transplantation [114].Such an approach would pose logistical challenges, including 1. a longer lead time for product requests as the product is not on the shelf; 2. The risk of unsuccessful or insufficient priming, which could delay product release; and 3. the need to study and validate the stability of primed MSCs before considering the transfer of the products to off-site clinical facilities.It will be interesting to investigate if IFNγ-primed MSCs can withstand cryopreservation and retain their immunomodulatory properties.If this is shown to be feasible, it will greatly ease the logistic issue of scheduling transplantation and reduce the cost of manufacturing.
Other than that, there are two ongoing phase 2 clinical trials initiated in 2020 and 2022 utilizing hypoxia-primed MSCs for severe COVID-19 and chronic lumbar disc disease (cLDD), respectively.The COVID-19 trial employs secretome from hypoxic MSCs, while the cLDD trial utilizes autologous bone marrow hypoxic MSCs.In a chronic lower back pain case report, researchers observed that intra-discal injection of autologous, hypoxically cultured bone marrow-derived MSCs demonstrated safety and feasibility in five patients diagnosed with degenerative disc disease.The BM-MSCs were cultured in 5% oxygen throughout the entire culturation process, and patients were followed up 4-6 years post-MSC infusion [115].Additionally, Putra et al. demonstrated that hypoxia-preconditioned MSCs and the secretome exhibit superior effects in treating various diseases, including acute renal failure [116], full-thickness-wound [117], and more.While these ongoing trials suggest promise for the priming strategy, its effectiveness in humans awaits the publication of clinical trial data.The specific method and duration of hypoxia priming were not disclosed in these trials, but they indicate the feasibility of upscaling hypoxic MSC production.
For MSC up-scaling, various bioreactors have been developed.Due to the anchorage nature of these cells, most bioreactors require the use of multiple culture containers in stacks (cell factories) or the use of cell microcarriers, scaffolds or hollow fibres to increase the surface area for cell attachment and proliferation [118][119][120].The GMP production of hypoxic MSCs would likely require the entire cell manufacturing process to be performed in hypoxic workstations, a solution already commercially available and expected to become a mainstay for GMP cell manufacturing facilities.Newer GMP facilities should adopt modular installations where a flexible layout and custom-made workstations can be accommodated.This allows change to be implemented easily as part of the evolving needs of the industry.The adoption of the two-step approach, as mentioned above, may work as well for hypoxia priming, i.e., firstly, the large-scale expansion of MSC followed by cryopreservation, and secondly, the priming of revived MSCs in a hypoxic chamber.Such an approach faces similar challenges as previously mentioned.
EVs are isolated from the conditioned medium of MSC cultures, with isolation techniques varying between laboratories.Solutions for handling and processing large volumes of liquid are already established in the pharmaceutical industry.Tangential flow filtration may be preferred due to its gentle processing [121].Regardless of the methods used, the stability and storage of EVs must be evaluated.Given the short half-life of EVs, freeze-drying using cryoprotectants like mannitol has been developed [122].

Ethical and Regulatory Considerations
Ethical concerns regarding MSCs and their EVs have largely been addressed by using discarded tissues such as umbilical cords.However, it is crucial to obtain written informed consent, ensure transparency about the use and handling of donated samples, and protect donor confidentiality at all times.The European Medicines Agency classifies MSCs and their EVs as Advanced Therapeutic Medicinal Products (ATMPs) (Advanced therapy medicinal products, https://www.ema.europa.eu/en/human-regulatoryoverview/advanced-therapy-medicinal-products-overview(accessed on 17 May 2024)), while the US Food and Drug Administration categorizes them as Cell & Gene Therapy Products (CGTPs) (Framework for the Regulation of Regenerative Medicine Products, https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/framework-regulation-regenerative-medicine-products (accessed on 17 May 2024)).While jurisdiction in emerging countries may still be in its infancy, most developing regulators will adopt the guidelines from these mature agencies.The primary concern in the regulation of medicinal products is safety besides their purity and quality.In general, the safety of these products after extensive expansion and manipulation must be assessed.In addition to incorporating a potency or functional assay, there is also a need to rule out tumorigenicity and abnormal karyotype for the MSCs.To minimize off-target effects, it is recommended to measure known markers for side effects, such as apoptotic or senescent markers and inflammatory markers, in MSCs and their EVs as exclusion release criteria.Such safety concerns may be heightened after cell priming due to the pre-stimulated state and increased potency of MSCs or their EVs.Since IFN-γ is a potent inducer of immune cell proliferation, the safety of its residual amount in primed MSCs and in the EV cargo that is administered should be established.A cut-off level for the residual IFN-γ and in the EV cargo needs to be ascertained before release.Additionally, it is essential to source clinical and GMP-grade IFN to ensure quality and consistency for priming.

Conclusions and Future Recommendations
In conclusion, the current review highlights the potential of IFN-γ and hypoxia priming, either singularly or in combination, to enhance the therapeutic effectiveness of MSCs in regenerative medicine.This enhancement occurs via the modulation of signalling pathways such as JAK/STAT and PI3K/AKT, and Leptin/JAK/STAT and TGF-β/Smad.IFN-γ and hypoxia priming of MSCs influence their extracellular vesicle cargo and show promise as a cell-free therapeutic approach.The upregulation of IDO expression, which further suppresses T-cell proliferation upon IFN-γ priming, along with the consistent expression of HIF-1 after hypoxia priming, suggest their potential as reliable in vitro markers for evaluating the efficacy of these priming methods.Therefore, we propose considering them as tests for developing a standardized protocol (Figure 3).However, no specific methodologies have yet demonstrated promising repeatability in EVs derived from primed MSCs.Presumably, the therapeutic effects of primed MSCs will be passed down to their EVs.However, quality control should be performed both at the EV cell source and the EVs.

Figure 3 .
Figure 3. Flow chart for the systematic approach to developing primed MSC and EV therapies from GMP to commercialization.A structured approach has been proposed for optimizing MSC priming conditions by systematically adjusting both the concentration and duration of the priming agents to enhance the therapeutic efficacy of MSCs and their EVs.Primed MSCs and their EVs must fulfill inclusion and exclusion release criteria before proceeding to efficacy and safety validation in preclinical studies.Following this, a first-in-human trial for safety and dosage escalation is conducted before entering Phase II, Phase III and Phase IV clinical trials.GMP, Good manufacturing practice; GLP, Good laboratory practice; NOD/SCID, Nonobese diabetic/severe combined immunodeficiency.Author Contributions: Conceptualization, Y.L.T., M.E.A.-M., J.X.L. and M.H.N.; Methodology, Y.L.T. and M.E.A.-M.; Writing-Original Draft, Y.L.T. and M.E.A.-M.; Writing-Review and Editing, S.P.E., M.N.S., J.X.L. and M.H.N.; Visualization, Y.L.T.; Supervision, M.N.S., J.X.L. and M.H.N.; Funding Acquisition, M.H.N.All authors have read and agreed to the published version of the manuscript.Funding: Fundamental Research Grants: FF-2023-114 and FF-2020-189 (Faculty of Medicine, Universiti Kebangsaan Malaysia).The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.Institutional Review Board Statement: Not applicable.

Figure 3 .
Figure 3. Flow chart for the systematic approach to developing primed MSC and EV therapies from GMP to commercialization.A structured approach has been proposed for optimizing MSC priming conditions by systematically adjusting both the concentration and duration of the priming agents to enhance the therapeutic efficacy of MSCs and their EVs.Primed MSCs and their EVs must fulfill inclusion and exclusion release criteria before proceeding to efficacy and safety validation in preclinical studies.Following this, a first-in-human trial for safety and dosage escalation is conducted before entering Phase II, Phase III and Phase IV clinical trials.GMP, Good manufacturing practice; GLP, Good laboratory practice; NOD/SCID, Nonobese diabetic/severe combined immunodeficiency.

Table 1 .
Pre-clinical studies reported priming with IFN-γ as a strategy for enhancing the therapeutic efficacy of MSC therapies.

Table 2 .
Pre-clinical studies reported priming with hypoxia as a strategy for enhancing the therapeutic efficacy of MSC therapies.