Bone marrow mesenchymal stem cell donors with a high body mass index display elevated endoplasmic reticulum stress and are functionally impaired

Bone marrow mesenchymal stem cells (BM‐MSCs) are promising candidates for regenerative medicine purposes. The effect of obesity on the function of BM‐MSCs is currently unknown. Here, we assessed how obesity affects the function of BM‐MSCs and the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) therein. BM‐MSCs were obtained from healthy donors with a normal (<25) or high (>30) body mass index (BMI). High‐BMI BM‐MSCs displayed severely impaired osteogenic and diminished adipogenic differentiation, decreased proliferation rates, increased senescence, and elevated expression of ER stress–related genes ATF4 and CHOP. Suppression of ER stress using tauroursodeoxycholic acid (TUDCA) and 4‐phenylbutyrate (4‐PBA) resulted in partial recovery of osteogenic differentiation capacity, with a significant increase in the expression of ALPL and improvement in the UPR. These data indicate that BMI is important during the selection of BM‐MSC donors for regenerative medicine purposes and that application of high‐BMI BM‐MSCs with TUDCA or 4‐PBA may improve stem cell function. However, whether this improvement can be translated into an in vivo clinical advantage remains to be assessed.

significant differences have been found in terms of immunophenotypic, differentiation potentials, transcryptomic analyzes, proteomics, and immunomodulatory properties (Strioga, Viswanathan, Darinskas, Slaby, & Michalek, 2012). The effect of obesity on the function of BM-MSCs is unknown, although an increase in BM adipocytes has been shown to negatively affect the BM niche (Adler, Kaushansky, & Rubin, 2014;Emmons et al., 2017), disturb cell-cell interactions, hematopoiesis, and bone regeneration (Ambrosi et al., 2017). Therefore, it is conceivable that BM-MSCs, similar to ADSCs, may lose their differentiation capacities and stemness as a result of obesity. This may have direct implications for the use of MSCs obtained from high-BMI donors in regenerative medicine. Accumulation of mis-or unfolded proteins in the lumen of the endoplasmic reticulum (ER) results in ER stress and initiates a self-rescue process, known as the unfolded protein response (UPR; Bravo et al., 2013). Under physiological conditions, the stress sensors inositolrequiring enzyme-1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) are linked to binding immunoglobulin protein (BiP)/78-kDa glucoseregulated protein (GRP78), a small chaperone molecule (J. Wu & Kaufman, 2006). During ER stress, BiP/GRP78 becomes separated from the sensors, IRE1α and PERK undergo transphosphorylation, ATF6 is transported to Golgi and cleaved, and various transcription factors (XBP1, ATF4, ATF6, NFκB, CHOP) and protein kinases (JNK, AKT) are stimulated, resulting in the activation of the UPR (Park & Ozcan, 2013) to restore homeostasis (Bravo et al., 2013). Each of these pathways yields a specialized response during differentiation and metabolic processes (J. Wu & Kaufman, 2006). Tauroursodeoxycholic acid (TUDCA) attenuates ER stress and prevents UPR dysfunction, and has been shown to regulate stem cell differentiation into adipogenic and osteogenic lineages (Cha et al., 2016(Cha et al., , 2014. Its main action is channeled through the PERK pathway, modulating ATF4 and CHOP expression (Malo et al., 2010). Sodium 4-phenylbutyrate (4-PBA) stabilizes protein conformation in the ER and represses UPR activation, and has been shown to inhibit adipogenesis by modulation of the UPR (Basseri, Lhotak, Sharma, & Austin, 2009). The effects of 4-PBA are mediated through the modulation of both PERK and IRE1α pathways (Kim et al., 2012).
In this study, we functionally compared BM-MSCs from high-BMI (>30) and normal-BMI (<25) donors, and assessed the levels of ER stress during expansion and differentiation. In addition, we investigated how suppression of ER stress, using TUDCA and 4-PBA, affected expansion and differentiation capacities of BM-MSCs from high-BMI donors. We hypothesized that the modulation of ER stress in high-BMI BM-MSCs could be used to produce better quality cellular products for regenerative medicine applications.
Cultures were maintained at 37 o C and 5% CO 2 , and medium was replaced every 3-4 days. Experiments were carried out with BM-MSCs at passage 3.  Stained cells were imaged using bright-field microscopy (Olympus, Tokyo, Japan) and counted using the ImageJ software.

| RT-PCR
To assess gene expression, total RNAs were extracted from BM-MSCs using the RNA Protect Cell Reagent and RNeasy ® Plus Mini Kit (Qiagen, Germany). RNA was converted into cDNA using the High-BMI BM-MSCs display reduced proliferation, decreased osteogenic differentiation, and increased senescence.

| Statistical analysis
Significant differences between the means of two groups were calculated using Student's t-test and variances between two groups were determined using F-tests in the GraphPad Prism software (www.graphpad.com/scientific-software/prism/).  For comparison, data from normal-BMI BM-MSCs were added. Data are given as mean + standard deviation. Treatment with TUDCA and 4-PBA improves both osteogenic and adipogenic differentiation. The F-test was used to compare variances between two groups, *p < 0.05; **p < 0.01; ***p < 0.001. BMI, body mass index; BM, bone marrow; MSCs, mesenchymal stem cells; 4-PBA, 4-phenylbutyrate; TUDCA, tauroursodeoxycholic acid; UPR, unfolded protein response BM-MSCs was increased at baseline, but rapidly decreased at Day 1 of adipogenic differentiation (p < 0.01).

| TUDCA and 4-PBA improve osteogenic differentiation and normalize the UPR of high-BMI BM-MSCs
Treatment of high-BMI BM-MSCs (n =6) with TUDCA or 4-PBA resulted in a significant increase in ALPL gene expression (both conditions p < 0.01) and partial recovery of osteogenic differentiation.
After osteogenic differentiation for 7 days in the presence of TUDCA, gene expression of ATF4 (p < 0.001), XBP1, and CHOP (both p < 0.05) increased significantly (Figure 3a), resembling the gene expression pattern during UPR of normal-BMI BM-MSCs more closely, whereas osteogenic differentiation in the presence of 4-PBA showed only a significant effect on ATF4 expression (p < 0.01). After adipogenic differentiation in the presence of TUDCA or 4-PBA for 7 days, gene expression of PPARG and ATF4 was found to be significantly upregulated (p < 0.05), whereas CHOP was markedly downregulated compared with cultures without chaperones (p < 0.0001).

| DISCUSSION
BM-MSCs have rapidly gained importance for their use in regenerative medicine due to their relatively easy collection and expansion procedures, and their wide differentiation potential (Malgieri et al., 2010;Nombela-Arrieta et al., 2011;Odabas et al., 2014). Maintenance of differentiation capacity and stemness of BM-MSCs is crucial for their clinical application. With the steadily increasing rates of obesity (BMI ≥ 30 kg/m 2 ) in almost all populations in recent years (Turconi & Hellas, 2007;WHO, 2000), the elucidation of the effects of obesity and its metabolism on stem cell functions has become equally more important. Obesity-related changes in metabolism affect the immune system, generate low-level chronic inflammation, cause impaired tissue healing, and most important of all, predispose to the development of many associated diseases, including Type 2 diabetes, cardiovascular disease, cancer, musculoskeletal diseases, and neurodegenerative diseases (Emanuela et al., 2012). Obesity negatively affects the function and maintenance of bone marrow hematopoietic stem cells and adipose stromal-derived stem cells (Ambrosi et al., 2017;Badimon & Cubedo, 2017;Baptista et al., 2009;de Girolamo, Lucarelli, et al., 2013;De Girolamo, Stanco, et al., 2013), and has a negative impact on the differentiation of BM-MSCs in mice (C. L. Wu, Diekman, et al., 2013 ).
An increase in mis-or unfolded proteins creates a stress condition in the ER called ER stress (Bravo et al., 2013). During physiological conditions, such as stem cell differentiation and proliferation, a certain level of ER stress may be required to compensate for increased cellular demands (Matsuzaki et al., 2015).
However, pathophysiological conditions, such as obesity and diabetes, may induce ER stress as well (Ariyasu, Yoshida, & Hasegawa, 2017;Januszyk et al., 2014;Pagliassotti, Kim, Estrada, Stewart, & Gentile, 2016) and studies in mouse models have shown that both of these metabolic conditions may negatively affect stem cell behavior (Januszyk et al., 2014;D. Wu, Ren, Pae, Han, & Meydani, 2013). The mechanisms through which obesity, in the absence of comorbidity, affects stem cell functions, and BM-MSCs in particular, are currently not fully understood, despite the presence of accumulating evidence indicating the importance of ER stress in the dysfunction of stem cells in obesity (Pagliassotti et al., 2016).
As a result of the increasing frequency of obesity in the general population, it is likely that a substantial fraction of future BM-MSC donors may be overweight or obese. This may have direct implications for their use, particularly in the field of regenerative medicine, where these cells could be potentially used for the treatment of orthopedic issues, such as critical-size bone fractures or cartilage defects. It is conceivable that BM-MSCs, ADSCs, or other stem cells, obtained from donors with advanced overweight, may display severe differentiation and proliferation defects, resulting in poor quality of the isolated stem cell product and decreased regenerative potential in vivo. Alleviation of ER stress, using a variety of ER stress inhibitors, including, but not restricted to, TUDCA and 4-PBA, could help improve the quality of the final stem cell product.
In this study, we found that baseline levels of ATF4 and CHOP were significantly increased in high-BMI BM-MSCs in comparison with normal-BMI controls. An increase in ATF4 and CHOP expression is typically seen after phosphorylation and activation of the PERK pathway and results in activation of the UPR. These data indicate that especially the PERK pathway appears to be activated in dealing with the negative results of obesity. Furthermore, the expressions of both ATF4 and CHOP should increase during normal osteogenic differentiation, but failed to do so in the high-BMI BM-MSC group. TUDCA has been shown to attenuate ER stress and prevent UPR dysfunction through prevention of Grp78/BiP upregulation, suppression of PERK and JNK phosphorylation, and prohibition of the expression of CHOP (Malo et al., 2010). In vitro, TUDCA was shown to enhance osteogenic differentiation of BM-MSCs (Cha et al., 2016;Han et al., 2013;Shi et al., 2017) and suppress the adipogenic differentiation of ADSCs (Cha et al., 2014).
Here, we found that although TUDCA was very effective in increasing osteogenic differentiation, and to a lesser extent adipogenic differentiation, its effects were mainly visible as a normalization of the UPR. stress through the regulation of several ER stress-inducible UPR-related proteins, including Grp78/BiP, Grp94, C/EBP homologous protein, eIF-2α, JNK, IRE-1α, PERK, and XBP-1 (Kim et al., 2012). Although 4-PBA was found to facilitate both osteogenic and adipogenic differentiation of BM-MSCs from high-BMI donors, the effects on ATF4 and CHOP were less robust. Since the administration of TUDCA and 4-PBA to high-BMI BM-MSCs resulted in an improvement in osteogenic differentiation and normalization of the UPR, and since 4-PBA and TUDCA modulate different UPR pathways, it is conceivable that optimized protocols, including both chaperones together or consecutively, might completely repair or at least further improve osteogenic differentiation. In addition, the fact that treatment with TUDCA and 4-PBA, which are known to modulate ER stress through suppression of the UPR, appear to partially correct the osteogenic differentiation of high-BMI BM-MSCs by increasing (normalizing), rather than decreasing, the expression of ATF4 and CHOP, suggests that TUDCA and 4-PBA may also act through other pathways.
In this study, we hypothesized that ER stress and the UPR might play a pivotal role in coping with cellular stress, and maintenance of the function and stemness of high-BMI BM-MSCs. We compared and