1. Introduction

The potential of adipose stromal/stem cell (ASC)-based therapies relies on their proliferation and differentiation capacity [1–3] as well as their secretory factors affecting angiogenesis, wound healing [4], cell survival, fibrosis, and neovascularization [5]. In particular, the immunomodulatory functions of ASCs have drawn attention to their utilization in cell-based therapies [6–8]. It has been demonstrated that ASCs secrete cytokines and exosomes that regulate the number and activity of immune cells [9, 10]. For example, ASCs can polarize proinflammatory (M1) macrophages toward anti-inflammatory (M2) macrophages [11–13] and modulate T-cell functions [14–16] in an inflammatory milieu.

Obesity-induced chronic low-grade inflammation impacts the physiology of adipose tissue [17] and disturbs the balance and functions of immune cells, such as T cells and macrophages, residing in adipose tissue [18]. M1 and M2 macrophages are abundant in adipose tissue, and their phenotypes are regulated by the surrounding environment [19]. In nonobese individuals, M2 macrophages maintain adipose tissue homeostasis, preventing inflammation and promoting insulin sensitivity [20], whereas M1 macrophages are associated with obesity-induced inflammation [21].

Obesity-induced inflammation not only interrupts the macrophage equilibrium in adipose tissue [22, 23] and accelerates the secretion of proinflammatory adipokines and cytokines [24] but also may affect ASC functions [25], such as their immunomodulatory capacity [16, 25, 26]. Compared with ASCs derived from normal-weight donors, human ASCs derived from obese donors (obASCs) exerted reduced immunosuppressive effects on mouse T cells in vitro [27], with similar effects on mouse macrophages in vivo [8, 28, 29]. In contrast, compared with obASCs, ASCs derived from donors of normal weight exerted strong immunosuppressive effects on the macrophage phenotype [30]. Additionally, a recently published study showed that ASCs derived from previously morbidly obese donors behaved similarly to ASCs derived from overweight donors, as both promote M2 macrophage polarization and exhibit a mixed pro- and anti-inflammatory secretome [31].

Bariatric surgery is an effective weight loss procedure [32] that may restore metabolic functions by reducing subcutaneous fatty acid uptake and positively affecting hepatokines and plasma inflammatory markers [22, 23]. While the majority of studies focusing on the effects of donor weight on ASC properties have compared ASCs derived from donors with either normal weight or obesity [8, 16, 25–30], only a few studies have focused on the effects of weight loss [31, 33–35]. Moreover, only a few studies have reported the effects of bariatric surgery-associated weight loss, including reduced DNA damage, improved viability, and decreased adipogenic differentiation of either ASCs or the stromal vascular fraction [33, 34]. Additionally, some studies have shown the effects of obesity, weight loss, and bariatric surgery-related weight loss on angiogenic [36] and pericyte functions [35]. However, some of these studies have not mentioned the weight loss methods employed [36]. Few studies have examined the clinical implications of weight loss-induced changes in ASCs despite their use in regenerative medicine and immunotherapy [31, 33–35]. For instance, Alves et al. reported that ASCs from ex-obese donors exhibit an altered immunomodulatory profile, polarizing M0 macrophages toward an M2-like phenotype with a mixed (M1/M2) secretory response. This suggests that weight loss history could influence ASC-based therapies for inflammatory diseases such as Crohn’s disease and spinal cord injury [31]. Similarly, Schmitz et al. [34] observed a negative correlation between donor weight loss history and ASC functional activities, including proliferation and migration capacity, indicating that ASCs from individuals who have lost weight may have diminished regenerative potential. In addition, Silva et al. found that weight loss following bariatric surgery alters ASC properties and increases pericyte function, monocyte chemoattractant protein-1 (MCP-1) secretion, and lipid accumulation, which could affect their regenerative potential. However, further molecular investigations are needed to ensure that autologous ASC therapies remain effective post-weight loss [35]. Despite these findings, there is a lack of follow-up in vitro and in vivo studies from the same individual before and after the weight loss investigating the long-term effects of bariatric surgery-induced weight loss on ASC function, particularly their immunomodulatory interactions with macrophages, which are key immune cells regulating adipose tissue homeostasis [37]. Understanding these changes is critical to optimizing ASC-based therapeutic applications and ensuring their efficacy in clinical settings. Therefore, this study aimed to analyze the effects of weight loss on the characteristics and immunomodulatory functions of ASCs derived from the same donors before and after bariatric surgery-associated weight loss.

In this study, ASCs were isolated before (obASCs) and after weight loss (wlASCs) from donors who underwent bariatric surgery, maintaining a similar genetic background before and after weight loss. Donor inflammatory status was examined by analyzing plasma C-reactive protein (CRP), leptin, and adiponectin concentrations. From adipose tissue histological samples, adipocyte size and the presence of crown-like structures (CLSs) were evaluated. The surface marker expression and differentiation capacity of obASCs and wlASCs were also analyzed. Moreover, the oxygen consumption rate (OCR) of ASCs was studied by examining mitochondrial functions. To evaluate the immunomodulatory capacity of ASCs, macrophages were polarized and activated toward the M1 and M2 phenotypes from peripheral blood mononuclear cells (PBMCs) and studied in direct cocultures with ASCs to mimic the environment of adipose tissue where cells are in contact with each other [22, 23]. Surface marker profiles, pro- and anti-inflammatory gene expression, and cytokine secretion in mono- and cocultures were assessed to study cell‒cell interactions.

2. Materials and Methods

3. Results

3.1. Decreased Plasma Levels of Inflammatory Markers After Weight Loss

Bariatric surgery has been demonstrated to facilitate weight loss and improve metabolic functions [32, 45–48]. In our study, we observed that the BMI of ASC donors (n = 6) significantly decreased after bariatric surgery (ΔBMI: average 15.41, p = 0.0313) (Table S6).

Previous reports have highlighted an imbalance of plasma pro- and anti-inflammatory markers associated with obesity and related metabolic disorders [49, 50]. To determine how weight loss impacts donor inflammatory status, we analyzed the adipokines leptin and adiponectin, as well as CRP, in blood plasma samples from donors before (n = 6) and after weight loss (n = 6). Our results aligned with those of previous studies showing improvements in plasma adipokines [24, 51] and CRP levels [52, 53] after weight loss. Our findings revealed a notable decrease in the inflammatory CRP level (Figure 1A), a significant decrease in the leptin level (p = 0.0312; Figure 1B), and a significant increase in the anti-inflammatory adiponectin level (p = 0.0312) following weight loss (Figure 1C).

3.3. Improved Mitochondrial Respiration Capacity of ASCs After Weight Loss

Obesity-induced inflammation has been demonstrated to induce oxidative stress in adipocytes. This stress impairs their mitochondrial functions and reduces the OCR of ASCs, especially compared to ASCs derived from individuals of normal weight [59–61]. However, bariatric surgery-related weight loss has been shown to increase mitochondrial respiration within adipose tissue [62]. To investigate this phenomenon further, we examined the overall mitochondrial respiration rates of obASCs (n = 6) and wlASCs (n = 6) by measuring the basal, maximal respiration, ATP production, proton leakage, and spare respiratory capacity of the cells. Although we did not observe significant differences when comparing individual factors (Figure 2B–F), consistent with previous findings, we found that the overall mitochondrial respiration rate over the 120-min measurement period was significantly greater in wlASCs (p  < 0.0001) than in obASCs (Figure 2A).

3.5. Pro- and Anti-Inflammatory Gene Expression and Cytokine Secretion in ASC Monocultures

3.5.1. Downregulation of Both Pro- and Anti-Inflammatory Gene Expression in ASCs After Weight Loss

ASCs are highly attractive for use in cellular therapies, particularly because of their immunomodulatory functions, in which cytokine secretion plays an important role [1]. However, Oñate et al. and Serena et al. previously explored the impact of weight loss on the gene expression of immunomodulatory factors in ASCs, and none have specifically investigated the same donor before and after weight loss [30, 69]. In this study, we examined the effects of weight loss on pro- and anti-inflammatory gene expression levels in ASC monocultures (Figure 3A–D and Figure S5). Our findings revealed significant downregulations in the expression of proinflammatory genes in wlASCs (n = 4) compared with obASCs (n = 4, TNF-α: p = 0.0329, chemokine ligand 5 [CCL5]: p = 0.0158, and cyclooxygenase-2 [COX2]: p = 0.0076; Figure 3A–C). Additionally, we observed a significant decrease in the expression of the anti-inflammatory gene tumor necrosis factor-inducible gene 6 (TSG6) in wlASCs (p = 0.0378) compared with obASCs (Figure 3D).

3.5.2. Decreased Secretion of the Proinflammatory Cytokine IL-12p70 by ASCs After Weight Loss

Obesity-induced inflammation alters cytokine secretion in adipose tissue [70], which changes the ASC cytokine profile to be more proinflammatory [35]. For example, obesity-induced inflammation elevates the levels of IL-12p70, a member of the IL-12 family, in both immune cells and adipocytes [71, 72]. However, cytokines of the IL-12 family have both proinflammatory and immunoregulatory functions [73, 74]. As reported by Verma et al. [75], IL-12p70 may also play a role in downregulating immune responses by inducing regulatory T cells. In this study, the overall secreted amounts of proinflammatory cytokines were low in both obASCs (n = 4) and wlASCs (n = 4) (Figure S6). However, compared with obASCs, wlASCs (p = 0.0409) secreted significantly less IL-12p70 (Figure 3E). Additionally, nonsignificant decreases in the levels of all the studied anti-inflammatory cytokines, such as IL-4, IL-1RA, and IL-10, were observed after weight loss (Figure S6G–I). In line with our results, Silva et al. [35] reported that weight loss did not fully improve cytokine secretion by ASCs.

3.6. Downregulation of Both Pro- and Anti-Inflammatory Surface Markers in M1 Macrophages by obASCs and wlASCs in Cocultures

Previous studies have indicated that the immunomodulatory capacity of ASCs is strongly linked to donor BMI. Specifically, conditioned media or exosomes from ASCs obtained from donors with normal weight have been shown to reduce inflammation and shift the macrophage phenotype from M1 to M2 both in vitro and in vivo [76–78]. However, ASCs derived from obese donors may promote M1 polarization [26] and exhibit a reduced or impaired ability to suppress M1 macrophages while activating M2 macrophages [26, 30]. In contrast to previous findings, our study revealed that ASCs did not direct M1 macrophages toward the M2 phenotype; that is, they did not function as anti-inflammatory cells. In our study, compared with those in M1 monocultures, downregulation of the proinflammatory cluster of differentiation (CD) markers CD11C and CD86 and the anti-inflammatory marker CD163 was observed in cocultures with both obASCs and wlASCs (Figure 4). Specifically, significant decreases in the percentages of CD11C (p = 0.0166)- and CD86 (p = 0.0386)-positive macrophages were observed in M1 cocultures with wlASCs compared with those in M1 monocultures (Figure 4A,C). Similarly, the percentage of CD163-positive macrophages was significantly lower in M1 cocultures with both obASCs (p = 0.0072) and wlASCs (p = 0.0009) than in M1 monocultures (Figure 4G).

3.8. Pro- and Anti-Inflammatory Gene Expression and Cytokine Secretion in M1 Macrophage Cocultures

3.8.1. Inconsistent Regulation of Pro- and Anti-Inflammatory Gene Expression in M1 Macrophages by obASCs and wlASCs in Cocultures

In contrast to our surface marker results, we observed significant decreases in the expression levels of proinflammatory genes, specifically IFNɣ and TNF-α, in macrophage cocultures with both obASCs and wlASCs (Figure 5). Compared with their corresponding macrophage monocultures, the M1 cocultures with obASCs and wlASCs (obASCs; p = 0.0151 and wlASCs; p = 0.0343) presented significantly lower IFNɣ expression (Figure 5A). Similarly, the expression of TNF-α was significantly lower in M1 cocultures (obASCs; p = 0.0058 and wlASCs; p = 0.0176) than in their respective macrophage monocultures (Figure 5B).

Interestingly, CCL5, which is associated with macrophage recruitment and M1 polarization during obesity-induced inflammation [79–81], exhibited a different pattern. In our study, the expression of CCL5 was significantly increased in M1 cocultures with both obASCs and wlASCs (obASCs; p = 0.0088 and wlASCs; p = 0.0025) (Figure 5C). This result contrasts with those of previous studies suggesting that ASCs possess anti-inflammatory abilities that downregulate CCL5 expression [82]. However, a recent study reported that ASC-mediated CCL5 plays a crucial role in T-cell accumulation within adipose tissue, antagonizing obesity-induced inflammation [83].

In our study, we observed a modest downregulation of anti-inflammatory gene expression in M1 cocultures with both obASCs and wlASCs (Figure 5 and Figure S14). Compared with M1 monocultures, M1 macrophages cocultured with wlASCs presented significantly lower mannose receptor C-type 1 (MRC1) expression (p = 0.0382) (Figure 5D). In M1 cocultures with obASCs, peroxisome proliferator-activated receptor-γ (PPARɣ) expression was significantly lower (p = 0.0068) than that in M1 monocultures (Figure 5F). Similarly, KLF-4 expression in M1 cocultures with obASCs was significantly lower (p = 0.0061) than that in M1 monocultures (Figure S14B). Furthermore, compared with M1 monocultures, M1 cocultures with obASCs presented significantly decreased human leukocyte antigen G (HLA-G) expression (Figure S14F).

3.8.2. Increased Secretion of Proinflammatory Cytokines by ASCs Before and After Weight Loss in M1 Cocultures

In M1 cocultures with both obASCs and wlASCs (Figure 5), there were notable increases in the secretion of the proinflammatory cytokines MCP-1, IL-6, IL-12p70, and IL-1β. IL-12p70 and IL-1β secretion levels were significantly greater, particularly in wlASCs, than in M1 monocultures. We observed significantly increased secretion of MCP-1 in M1 cocultures with obASCs (p = 0.0242) compared with M1 monocultures (Figure 5G). These results align with previous findings by Silva et al. [35], where MCP-1 secretion was notably greater in M1 cocultures with obASCs than in those with ASCs from normal-weight donors. Similarly, the secretion levels of IL-12p70 (p = 0.0257, Figure 5J) and IL-1β (p = 0.0352, Figure S14H) were significantly greater in M1 cocultures with wlASCs than in M1 monocultures. In contrast, a significant decrease in the secretion of the proinflammatory chemokine MDC was observed in M1 cocultures with ASCs (obASCs; p = 0.0166 and wlASCs; p = 0.0388) compared with M1 monocultures (Figure 5I).

The secretion levels of IL-6 and IL-4 were significantly greater in both obASC (IL-6; p = 0.0098 and IL-4; p = 0.0266) and wlASC (IL-6; p = 0.0024 and IL-4; p = 0.0025) cocultures than in M1 monocultures (Figure 5H, L, respectively). Previous studies have highlighted the pleiotropic role of IL-6 secretion by MSCs in mediating immunomodulatory responses, particularly during inflammation [16, 84, 85]. Interestingly, an in vivo mouse study reported that IL-6 secretion from adipocytes derived from donors with obesity could stimulate adipose tissue macrophages, leading to IL-4 signaling and contributing to an anti-inflammatory environment [86]. In our study, we observed simultaneous increases in both IL-6 and IL-4 secretion. Therefore, we speculate that the increased secretion of IL-6 plays a role in the concurrent increase in IL-4 secretion in the cocultures.

3.9. Pro- and Anti-Inflammatory Gene Expression and Cytokine Secretion in M2 Macrophage Cocultures

3.9.1. Inconsistent Regulation of Pro- and Anti-Inflammatory Gene Expression and Cytokine Secretion in M2 Macrophages by obASCs and wlASCs in Cocultures

With respect to the M2 cocultures, we did not observe any significant differences in proinflammatory gene expression (Figure 6A–C). Like in M1 cocultures, the expression of anti-inflammatory genes was slightly downregulated in M2 cocultures with both obASCs and wlASCs (Figure 6 and Figure S15). Notably, TSG6, a protein secreted by ASCs, plays a crucial role in regulating macrophage function and reprograms them toward an anti-inflammatory phenotype when in contact with ASCs [87–91]. In line with these findings, we observed that TSG6 expression was significantly greater in M2 cocultures with ASCs (obASCs; p = 0.0197 and wlASCs; p = 0.0172) than in M2 monocultures. Additionally, TSG6 expression was slightly but significantly greater in M2 cocultures with wlASCs (p = 0.0315) than in M2 cocultures with obASCs (Figure 6E). In an inflammatory environment, MSC expression of TSG6 increases, especially in contact with macrophages, leading to attenuated inflammation and enhanced tissue repair [87, 92–96].

PPARɣ, a critical player in macrophage polarization toward the M2 phenotype, serves as a key regulator of the inflammatory potential of tissue macrophages [97]. Moreover, in AT, obesity-induced inflammation downregulates PPARɣ expression in M2 macrophages, exacerbating the inflammatory milieu [98]. Restoring PPARɣ expression is essential for reinstating the anti-inflammatory functions of both macrophages and adipocytes, which become compromised during obesity-induced inflammation [99]. Surprisingly, contrary to previous findings, our study revealed that weight loss did not lead to the restoration of PPARɣ expression. Notably, in M2 cocultures with wlASCs, PPARɣ expression was significantly lower (p = 0.0157) than that in M2 cocultures with obASCs (Figure 6F). Consistent with PPARɣ expression, M2 cocultures with wlASCs presented markedly reduced levels of the anti-inflammatory protein HLA-G (Figure S15F), the secretion of which has been reported to enhance anti-inflammatory and immunosuppressive responses in an inflammatory environment [100].

3.9.2. Similar Regulation of Cytokine Secretion by ASCs Before and After Weight Loss in M2 Cocultures

Like M1 cocultures, M2 cocultures with wlASCs (p = 0.0464) exhibited significantly reduced secretion of the proinflammatory chemokine MDC compared with their respective monocultures (Figure 6I). Furthermore, increased secretion levels of the proinflammatory cytokines IL-6 and IL-12p70 were observed in M2 cocultures with both obASCs and wlASCs (Figure 6). The secretion of IL-6 was significantly greater in M2 cocultures with wlASCs (wlASCs; p = 0.0038) than in M2 monocultures (Figure 6H). Similarly, the secretion of IL-12p70 was significantly greater in M2 cocultures with obASCs (obASCs; p = 0.0161) than in M2 monocultures (Figure 6J). In contrast to M1 cocultures, significantly increased secretion of anti-inflammatory IL-1RA was observed in M2 cocultures with obASCs (p = 0.0350) compared with M2 cocultures with wlASCs (Figure 6K). These findings diverge from those of previous studies where ASCs derived from nonobese donors (BMI < 30 kg/m²) not only polarized M1 macrophages toward M2 macrophages but also enhanced the anti-inflammatory properties of M2 macrophages when cultured in ASC-conditioned medium [77]. Notably, IL-4 quantification for M2 macrophages was not included in the results, as IL-4 was utilized for their polarization and activation.

4. Discussion

Human ASCs are appealing candidates for cell-based therapies [1–4]. However, certain properties of ASC donors, such as obesity, have been found to negatively impact ASC characteristics and their immunomodulatory functions [16, 26, 63], thereby limiting their clinical potential. However, weight loss has been shown to restore individuals’ metabolic health [45, 47, 48] and improve ASC properties [33–36].

Consistent with previous studies, in our study, we observed that weight loss led to improved plasma inflammatory marker levels and reduced adipose tissue inflammation. Additionally, we detected changes in ASC characteristics or functions, such as mitochondrial respiration, the CD marker profile, proliferation, and adipogenesis, in accordance with previous findings [52, 58, 60, 63].

In line with Onate et al. and Serena et al. [30, 69] in wlASC monocultures, we observed significant downregulations in the expression of all studied proinflammatory genes. Additionally, the secretion of the proinflammatory cytokine IL-12p70 was reduced in ASCs following weight loss. However, contrary to earlier findings, the expression of the anti-inflammatory gene TSG6 was also significantly downregulated in our study. Notably, La Rusa et al. reported that TSG6, which is secreted by MSCs, plays preventive and immunomodulative roles in various inflammatory diseases [90].

In contrast to prior studies [76–78], neither obASCs nor wlASCs directed inflammatory M1 macrophages toward an anti-inflammatory M2 phenotype. Interestingly, earlier research suggested that ASCs guide primary human adipose tissue-derived M0 macrophages toward the M2 phenotype [30]. Based on these findings, we hypothesized that M2 macrophages would either maintain or enhance their anti-inflammatory phenotype when cocultured, especially with wlASCs. In contrast, our observations revealed that M2 macrophages were repolarized toward a proinflammatory phenotype in coculture with both obASCs and wlASCs.

While numerous of our macrophage coculture results suggest increased proinflammatory and decreased immunosuppressive properties with both obASCs and wlASCs, there are notable exceptions that may reflect the transitional state of these cells. For example, in M1 cocultures with wlASCs, we observed significant downregulation of the proinflammatory markers CD11C and CD86. Similarly, the secretion of the proinflammatory chemokine MDC was significantly reduced in cocultures of M1 macrophages with both obASCs and wlASCs, and in M2 macrophage cocultures with wlASCs, whereas the secretion of the anti-inflammatory cytokine IL-4 was significantly increased in M1 cocultures with both obASCs and wlASCs. These results partially align with a study by Lopes-Alves et al., where ASCs obtained from previously obese donors were able to induce the M2 phenotype, although their secretory profile was a mix of M1 and M2 [31]. Despite significant weight loss, our ASC donors remained overweight or obese, which could account for the conflicting results compared with those obtained with ASCs derived from normal-weight donors. Additionally, in our study, ASCs were obtained from the same donors before and after bariatric surgery-related weight loss, which is a unique study setting that has not been published previously.

The divergences in experimental designs may also play a role in the conflicting results obtained in different studies. For example, in contrast to previous work by Serena et al. [30], where human primary adipose tissue-derived M0 macrophages were cultured in an ASC-conditioned medium, our study focused on coculturing obASCs and wlASCs with prepolarized and preactivated human M1 and M2 macrophages derived from PBMCs. This approach aims to mimic the inflammatory environment of adipose tissue, where macrophages exist in varying states of inflammation [22, 23]. Wang et al. [61] had similar study settings in terms of macrophage polarization as those used in our study. However, they studied the paracrine effects of human ASCs from normal-weight donors versus obese donors on mouse macrophages and reported increased expression of proinflammatory M1 markers when cocultured with obASCs but no changes in M2-related markers [61]. Similarly, Harrison et al. [26] reported reduced immunomodulatory effects of human obASCs on murine macrophages and microglia with upregulated expression of proinflammatory genes compared with those of normal-weight donor-derived ASCs. In our study, coculture time points were chosen based on previous studies on the immunomodulatory properties of MSCs to analyze MSC-macrophage interactions [26, 42, 61]. However, variations in coculture duration may influence the results and should be considered when interpreting the findings.

Furthermore, in our study, after isolation from adipose tissue, ASCs were cultured in a medium containing human serum rather than FBS, which has been utilized in other studies [26, 30]. However, in our study, FBS was also utilized in cocultures with macrophages. In our preliminary experiments, the macrophages were not viable in a medium containing human serum; therefore, we cocultured the ASCs and macrophages in a medium containing FBS. In addition, the sample collection time after weight loss might have affected the results, as there might be changes in the weight and metabolic functions of the donors over the weight loss period. However, our schedule of obtaining samples after bariatric surgery was quite similar to that used in a previous study; for example, 2–4 years resulted in similar cytokine profiles [35]. In line with the findings of Silva et al., the cytokine profile of ASCs in our study did not fully recover after weight loss.

In this study, our aim was to compare the effects of weight loss on the characteristics and functions of ASCs obtained from the same donors before and after weight loss, which led to a small sample size. The small sample size of the groups is a study limitation; larger study groups may be needed to confirm our results. In addition, weight loss was facilitated by bariatric surgery, which might have affected the results. Bariatric surgery-related weight loss impacts adipose tissue physiology [57, 101]. However, the current literature does not provide comparisons between weight loss methods, that is, weight loss driven by lifestyle changes such as diet and exercise or the use of drugs such as semaglutide and how these methods affect ASCs. Furthermore, most of our ASC donors were females, presumably of Caucasian origin and within the same age range of 39–53 years; hence, more samples representing both sexes, different ethnicities, and wider age ranges might be needed to reveal their concomitant effects on weight loss. Importantly, despite experiencing considerable weight loss, all the donors were still either overweight or obese. Hence, a study group representing individuals with constant normal weight and a group that has reached normal weight after weight loss should be included in future studies to draw more definite conclusions about the effects of weight loss on ASC characteristics and functions.

The selection of donors for ASC-based allogeneic therapies should be guided by a comprehensive assessment of donor health, as various biological and lifestyle factors influence ASC function. Previous studies have demonstrated that in addition to donor obesity [16, 25–27] and weight loss [31, 34, 35], donor age [102–104], gender [105, 106], disease conditions [30, 107], and lifestyle factors [108] can impact the regenerative capacity and therapeutic potential of ASCs. Among these factors, BMI and weight loss history are particularly relevant, as they are associated with functional changes in ASCs that may influence their immunomodulatory properties and regenerative efficacy. However, BMI and weight loss history should not be considered in isolation but rather as part of a broader donor profiling strategy that integrates multiple biological and metabolic parameters. For instance, assessing inflammatory markers, metabolic health, or ASC functional assays could provide a more precise evaluation of donor suitability for ASC-based therapies.

5. Conclusions

In our study, in individuals with obesity, inflammation within adipose tissue strongly affected ASCs, influencing their gene expression and cytokine secretion and impairing their anti-inflammatory functions.

Bariatric surgery led to a significant reduction in BMI and improved individuals’ metabolic health. This approach alters the composition of adipose tissue and impacts adipose tissue-resident ASCs by increasing their overall cellular respiration and proliferation capacity while decreasing their ability to differentiate into adipocytes.

Our study underscores the importance of examining multiple pro- and anti-inflammatory factors when investigating the impact of donor weight on the immunomodulatory functions of ASCs. The impact of weight loss on the immunomodulatory functions of ASCs varied according to the markers analyzed. Following weight loss, ASCs exhibited increased proinflammatory properties when cocultured with macrophages, characterized by the downregulation of anti-inflammatory factors, along with the upregulation of several proinflammatory factors, compared with those in monocultures. Conversely, wlASCs also demonstrated improved immunosuppressive functions in coculture with macrophages, as indicated by the upregulation of TSG6 gene expression and IL-4 secretion. We could assume that, as the ASC donors are still overweight, ASCs have not resumed their normal functions and have mixed immunomodulatory behavior. Thus, despite extensive weight loss enhancing ASC metabolism, it remains insufficient to fully restore the immunosuppressive functions of ASCs.

To enhance clinical decision-making, further in vivo studies are needed to determine how donor characteristics, including BMI and weight loss history, translate into therapeutic outcomes. Establishing standardized criteria for ASC donor selection based on functional rather than solely anthropometric parameters will be essential for optimizing the safety and efficacy of ASC-based interventions.

Ethics Statement

The project “The effect of overweight and smoking on the biological properties of adipose tissue stem cells and the regulation of the immune response” was approved by the Ethics Committee of the Expert Responsibility area of Tampere University Hospital with approval number R16036 on 5.4.2016 and an extension provided on 19.6.2019.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Amna Adnan: conception, experimental design, data collection and interpretation, manuscript writing, and final approval of manuscript. Miia Juntunen: isolating ASCs, preparation and phenotype characterization of ASCs, data collection, manuscript writing, and final approval of manuscript. Tuula Tyrväinen: provision of adipose tissue specimen before weight loss and final approval of manuscript. Minna Kelloniemi: provision of adipose tissue specimen after weight loss and final approval of manuscript. Laura Kummola: supervising flow cytometer experiments, data interpretation, manuscript revision, and final approval of manuscript. Reija Autio: supervising the statistical analysis, data interpretation, manuscript revision, and final approval of manuscript. Mimmi Patrikoski: conception and design, financial support, manuscript writing, and final approval of manuscript. Susanna Miettinen: conception and design, financial support, manuscript writing, and final approval of the manuscript.

Funding

This work was supported by The Research Council of Finland (decisions 336666, 326588, 312413, 353177, and 356966 for Susanna Miettinen), State Funding for University-Level Health Research, Tampere University Hospital, Wellbeing Services County of Pirkanmaa (Susanna Miettinen), Academy of Finland (decision 311084 for Mimmi Patrikoski), Finnish Cultural Foundation (Miia Juntunen and Mimmi Patrikoski), and the Tampere University Doctoral Programme in Medicine, Biosciences and Biomedical Engineering (Amna Adnan and Miia Juntunen).

Supporting Information

Additional supporting information can be found online in the Supporting Information section.