Mesenchymal stem cells (MSCs) are non-hematopoietic cells capable of self-renewal and differentiation into cells of mesodermal origin. They are easily isolated from several tissues/organs and can be largely expanded under culture conditions. According to the International Society for Cell and Gene Therapy (ISCT), three minimal criteria were proposed for defining MSCs: 1) adherence to plastic; 2) surface antigen expression where MSC must express CD90, CD73, and CD105 and no expression for CD45, CD34, CD14 or CD11b, CD19 or CD79α, and HLA-DR; 3) multipotent differentiation potential where MSCs should be able to differentiate in vitro into osteoblasts, adipocytes, and chondroblasts (Dominici et al., 2006).

MSCs are being widely used as a source of adult stem cells for therapy of several diseases such as graft versus host disease (Le Blanc et al., 2004), Crohn’s disease (García-Olmo et al., 2005), multiple sclerosis (Connick et al., 2012), and COVID-19 (Shi et al., 2021). Most clinical protocols use high cell doses per patient which has an impact on MSC culture due to the requirement of a high replicative rate and numerous cell passages to achieve the desired dose.

MSCs have a limited lifespan in vitro and, after some cell divisions, they enter a state of senescence that is characterized by irregular cell morphology and a decrease in proliferation rate (Wagner et al., 2008). However, a senescence state could also be achieved in initial passages; senescence in dividing cells after a period of normal growth is followed by a proliferation cease. This phenomenon is accompanied by significant cellular changes such as the typical Hayflick phenomenon, a decrease in proliferation, telomere shortening, and impairment of functional properties (Bonab et al., 2006). The typical Hayflick phenomenon is a concept that helps explain some events that lead to aging of normal cells (Shay and Wright, 2000).

This study discusses a case where bone marrow-derived MSCs from a patient enrolled in a clinical protocol decreased their proliferation rate in an initial passage and the impact on the quality and number of cells intended to be delivered to the patient.

The patient’s bone marrow-derived MSCs were grown as described in Section 2.2. The patient, female, 60 years old, was included in the clinical trial due to urinary stress incontinence. In the protocol, it was established that MSCs should be cultivated with AS up to passage five in order to achieve 10 million cells to be infused after releasing tests were performed according to local guidelines (Table 3). According to the National Health Surveillance Agency (ANVISA) guidelines (RDC 508/2021), some guidelines should be followed in order to release cells for clinical use: microbiological examinations, endotoxin assay, mycoplasma detection by RT-PCR, karyotyping, immunophenotyping, and potency assay (differentiation).

Cells from patients 1, 2, and 3 stained positively for canonical MSC surface markers CD90, CD105, and CD73, whereas they were negative for CD45, CD19, CD14, and HLA-DR (Figure 1). However, #1 cells presented a decrease in CD105 expression where there was a negative population for this marker; 71.34% of CD105 expression for #1 patient when compared to #3 patient who presented 99.35%. Patient #2 also presented a decrease in CD105 expression (80.83 vs. 99.35%) (Table 4). The expression of CD34 was increased in #1 cells (15.84%) in relation to #2 (0.61%) and #3 (1.05%) (Table 4).

In this case report, MSC culture supplemented with 10% AS presented a senescence state at passage 3. According to Hayflick and Moorhead (1961), cells enter a state called replicative senescence after some passages, being they stop dividing the main characteristic. As observed in our case, the cumulative population doubling level (CPDL) was calculated according to the following formula: log₁₀ (N/N₀) x 3.33, where N is the number of cells at the harvesting and N₀ is the number of cells plated (Table 5). MSCs from the patients #1, #2, and #3 were cultivated according to a standard operation procedure and CPDL calculated. As observed in Figure 2, #1 (Figure 2A), #2 (Figure 2B), and #3 (Figure 2C), patients’ MSCs showed the same morphology; however, senescence changed the cell proliferation rate as seen on graphic (Figure 2D). The CPDL showed a logarithmic rate in #2 and #3 patient’s cells and at the same time, patient #1 showed a more linear rate. Table 5 shows the cell density plated in the different cell cultures and in the different passages.

To find out the explanation about cell culture low rate growth, MSCs from patient #1 were also cultivated with 10% fetal bovine serum (FBS) used as standard. Additionally, MSCs from #3 patient were also cultured with 10% patient’s AS and 10% FBS up to passage 3. MSCs were then submitted to senescence assay, using a senescence β-galactosidase staining kit, and data were analyzed after 24 and 48 h. Figure 3 represents the senescent cells from both patients under 10% FBS and/or 10% AS conditions. The upper panel shows the MSC from #3 patient with a reduced number of senescent cells in comparison to the lower panel (#1 MSCs). The senescent profile was more pronounced under AS supplementation after 48 h in #1 patient’s cells in comparison to #3 MSCs (p = 0.0019) (Figure 3). However, #1 patient’s MSCs cultivated with FBS also presented senescence cells, suggesting that this event is more related to a cell factor instead to some soluble molecule.

Surprisingly, #1 MSC differentiation into adipocytes and chondrocytes seemed to be normal (Figure 4A), but when analyzing the osteocyte differentiation, the staining seemed to be more diffuse when than that of #2 or #3 patient (Figure 4A). In the osteogenic differentiation, there is a non-specific staining and background in #1 patient; this may indicate that the cells are still at the beginning of osteogenic differentiation or the differentiation capacity is decreased. A better resolution image was obtained for the osteogenic differentiation from patients #1 and #3; as observed in Figure 4B, the osteogenic differentiation and staining with Alizarin red S showed more localized calcium deposits in patient #3 and also formed structures similar to lines; #1 cells showed calcium deposits more disorganized, without defined structures.

To quantitatively compare the cells from patients #1 and #3, qRT-PCR was used to detect the expression of genes related to pluripotency. The attenuated cell proliferation from patient #1 cells was further evidenced by increased expression of KLF4 (p = 0.0016) and reduced expression of SOX2 (p = 0.0014), OCT3.4, and NANOG (p = 0.0016) (Figure 5).

Southern blot was performed to measure telomere length in the patient’s MSCs #1, #3, #5, and #7. Patients #1, #2, and #4 were enrolled in the same clinical trial for urinary incontinence. In this assay, the patients were nominated as patient 1 (#1), patient 2 (#3), patient 3 (#4), and healthy donor.

It is well known that MSCs have longer telomeres than leukocytes (Baxter et al., 2004; Fathi et al., 2019). Although we do not have a control curve for MSC, we purposely plotted the MSC data on the leukocyte curve of healthy donors (Figure 6). We found that the healthy MSC donor had a longer telomere (9.2 Kb; 90^th percentile) than the telomere of patients 1 and 2 (7.1 Kb and 7.4 Kb, respectively; 50^th percentile), and patient 3 presented a telomere length comparable to the healthy donor (8.7 Kb; 90^th percentile) (Table 6). It is worth noting that the patients were close in age and the control was younger. Therefore, we found that MSC from patients 1 and 2 have shorter telomeres than patient 3 and healthy donor.

Senescence is a complex process that may require different approaches to alleviate or even prevent this profile and improve the clinical application of MSCs. In this study, we presented data from a single patient with uncommon MSC senescence at passage 3. Banfi et al. (2002) showed that bone marrow stromal cells display a decrease in telomere length when in culture, and despite their inability to maintain the telomere length (no telomerase activity was detected), these cells were able to undergo around 40 population doublings. Telomere shortening is observed over a lifetime in MSCs. The average telomere length in early-passage MSCs depends on the age of the donor, and it has been proposed that it ranges from 10 to 11 kb in cells from fetal to 7 kb in cells from postnatal (Guillot et al., 2007). Telomere-dependent MSC senescence has been confirmed; the growth stops when telomeres reach a length of 5.8–10.5 kb (Baxter et al., 2004). Although telomere shortening is a marker of cell senescence, as we have shown here, the length of telomeres varies from donor to donor, making telomere length an inconstant measure of MSC senescence (Li et al., 2017).

The differentiation capacity of stem cells is an important characteristic for the repair and regeneration of tissues/organs; however, the patient’s MSC differentiation into adipocytes and chondrocytes seemed to be normal, but the osteocyte differentiation may be reduced. Studies have been reported that the osteogenic activity of MSC is progressively reduced as a function of increasing lifespan (Banfi et al., 2000; Turinetto et al., 2016).

According to Martin et al. (1997), bone marrow stromal cells when cultivated using fibroblast growth factor-2 (FGF-2) were able to maintain their osteogenic potential after the in vitro expansion. However, Hagmann et al. (2013) showed that while maintaining its differentiation potential, the use of FGF-2 pre-selects some MSC phenotypes. Regarding the use of MSC in clinical protocols, the use of growth factors may not be the best option; some MSC subtypes can also account for its efficacy.

CD105 is involved in multiple functions of MSCs such as differentiation, angiogenesis, and regenerative potential (Aslan et al., 2006; Mark et al., 2013). Na et al. (2015) showed that the marker CD105 seems to be more sensitive than the others, CD73 and CD90, to viability-associated events, and CD105 may be used as an indicator of cell death. In addition, it can be an important marker to understand the mechanisms during MSC senescence. Corroborating our study, this marker was evaluated in the senescent MSC-derived microvesicles, and the results showed that senescent MSCs secreted microvesicles of smaller size, and the level of CD105⁺ also decreased (Lei et al., 2017). The senescence biomarker SA-β-gal is used to investigate the functional implications of aging in MSCs (Dimri et al., 1995). In this study, patients’ MSCs showed that a higher number of cells stained positive for SA-β-gal than healthy donors’ MSCs, especially after using AS as a supplement. Several studies have shown the importance of stem cells in repair and in regenerating tissue and organs. Differentiation capacity of young MSCs is superior to old cells, and old MSCs proliferated more slowly and became senescent due to telomere attrition (Kretlow et al., 2008; Li et al., 2017).

The molecule CD34 is a hematopoietic progenitor marker, and its expression is lost when the maturation process takes part (Hughes et al., 2020). The expression of CD34 is higher in vivo than in cultured MSCs, and some growth factors in culture medium could impact in its expression in vitro (Bellagamba et al., 2018; Viswanathan et al., 2019). An study showed a decrease in the proliferation rate in adventitial stromal cell-like cells, and it was related to the accumulation of CD105⁺/CD146⁺/CD271⁺ and also CD34⁺, which could represent a population that entered the cell cycle but did not complete the cell division (Braun et al., 2013). Patient #1 displayed a higher expression of CD34, which is not expected and could be related to an accumulation of non-proliferating cells, leading to the senescence state.

To evaluate the potential impairment of self-renewal and differentiation of MSC due to early senescence, we investigated the expression of genes related to pluripotency (KLF4, OCT 3.4, NANOG, and SOX2). An increased expression of KLF4 was observed in the patient’s MSC culture in comparison to healthy controls.

The zinc finger protein Krüppel-like factor 4 (KLF4) is responsible for regulating gene transcription and cell fate, promoting malignant transformation, cell differentiation, tumor suppression, and stem cell properties (Kaczynski et al., 2003; Rowland and Peeper, 2006; Lin et al., 2011).

KLF4 is known to act in different ways by reducing proliferation rates by pathways such as p21^Waf1/Cip1 and may be present in promoting malignant properties by suppressing p53 or by upregulation of NOTCH1 (Kaczynski et al., 2003; Lin et al., 2011). Direct blockage of KLF4 expression has been demonstrated to promote cell differentiation and reduce cell aging in MSCs (Rowland and Peeper, 2006). KLF4, a downstream mediator, can either repress or activate transcription and participate in cell cycle regulation and differentiation (Rowland et al., 2005; Li et al., 2013; Lu et al., 2019).

A decreased expression of NANOG, SOX2, and OCT3.4 was also observed in the patient’s MSC in comparison to healthy controls. The reduced expression of these genes may indicate a reduced proliferation rate in the patient’s MSCs in comparison to healthy control MSCs.

NANOG is a transcription factor that is involved in the self-renewal of embryonic stem cells. The knockdown of NANOG and/or OCT4 in MSCs has been shown to not maintain MSCs in an undifferentiated state. In contrast, the overexpression of OCT4 and NANOG in MSCs has been shown to increase the cell proliferation rate and differentiation potential and inhibit spontaneous differentiation, becoming essential markers for maintaining MSC properties (Shields et al., 1996).

POU5F1 (also known as OCT4 and OCT3.4) and SOX2 are essential transcription factors for pluripotency and self-renewal. Both genes are expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. The overexpression of OCT4/SOX2 in MSC has been shown to improve cell proliferation and differentiation. Many researchers have investigated the effects of these pluripotent genes on MSCs, but the results are still debatable. Forced expression of pluripotent cell-specific factors (Oct4, Sox2, Nanog, and cMyc) or combinations of these genes for reprogramming somatic or adult stem cells has been used to improve the pluripotency of MSCs (Zhang et al., 2000; Seo et al., 2009; Lin et al., 2011; Yoon et al., 2011; Han et al., 2014; Gawlik-Rzemieniewska et al., 2016). SOX2, implicated in the maintaining the stem cell potency of BM-MSCs (Otsubo et al., 2011), recently was reported in a senescence context; the co-culture of senescent endothelial cells with BM-MSCs was associated with an increased expression of miR-126a-3p, in association with a significant decrease of SOX2, targeted by miR-126a-3p (Lazzarini et al., 2019).

A previous study with BM-MSCs from different healthy young donors identified some markers that could predict the expansion capacity of MSCs before reaching senescence. In this study, for each early passage, the gene expression levels of pluripotency markers OCT4, NANOG, and SOX2 were correlated with the final population doubling (PD) number and, in agreement with our results, revealed that a high OCT4 gene expression might be a potential hallmark and predictor of MSCs’ lifespan in vitro (Piccinato et al., 2015). A recent study also showed that OCT4 maintains the self-renewal ability of MSCs and may reverse the senescence phenotype (Lu et al., 2019).

Low-passage cultures are recommended for clinical expansion of cultures (Lechanteur et al., 2016). Therefore, there is no explicit passage number provided for the 15 MSC products in clinical use, as resumed by Liu and contributors (2020). However, for MSCs to be clinically functional, it is essential to monitor the senescence of MSC aging. Recent studies have discussed and summarized some strategies for monitoring senescence, and the molecular mechanisms involved (Li et al., 2017; Liu and Chen, 2020). Although the topic has much clinical relevance, the current knowledge of senescence is mainly based on classical events, and sometimes, there are difficulties not previously described.

Further research is required to determine the approaches for safety and effectiveness in MSC-based cell therapy. MSC culture is quite simple; however, it is necessary to carefully monitor the expansion process to detect any potential changes that may interfere with clinical management. Improving the conditions for ex vivo expansion and monitoring of aging markers for MSCs would help suppress and monitor in vitro aging.