1. Introduction

Mesenchymal stem cells (MSCs) are a type of adult stem cells characterized by self-renewal and multidirectional differentiation potential. They primarily reside in connective tissues and organ mesenchyme. Current research focuses on MSCs derived from bone marrow, umbilical cord blood, peripheral blood, and adipose tissue. Studies have shown that MSCs exhibit characteristics such as cytokine secretion and microvesicle production, which are associated with their immunomodulatory effects [1,2]. Due to advantages including diverse sources, ease of isolation and culture, absence of ethical restrictions, and retention of stem cell properties through multiple passages, MSCs have emerged as ideal seed cells. Combined with scaffold materials and cytokines, they are now employed in cell and gene therapies for tissue repair and organ regeneration [3,4].

Under appropriate in vitro culture conditions, MSCs can be directionally induced to differentiate into adipocytes, osteoblasts, chondrocytes, and other cell types. While traditional in vitro culture typically occurs under normoxic conditions (20% oxygen concentration), the physiological and pathological environments often present hypoxic conditions. For instance, oxygen levels range from 10%-13% in human blood, 3%-9% in normal tissues/interstitial spaces, and 1%-7% in bone marrow cavities [5-7]. Oxygen significantly influences cellular proliferation and differentiation, as insufficient oxygen supply during in vitro culture may lead to cell death. However, studies modifying oxygen concentrations revealed that while MSCs are sensitive to ischemic/hypoxic conditions, hypoxia alone minimally induces apoptosis. When injected into ventricles of myocardial infarction mice, most MSCs died within 4 days under ischemic/hypoxic conditions [8]. Ferreira et al. reported that MSCs cultured under hypoxia alone for 48 h remained viable, but combined hypoxic and prolonged serum-free conditions caused significant cell death [9]. Thus, hypoxic conditions may not inherently disadvantage cell culture [9-10]. Consequently, modifying in vitro culture microenvironments to better mimic physiological or pathological hypoxic conditions has become an important research focus.

Current clinical applications of MSC-based transplantation face critical challenges. After migrating to lesion sites, MSCs exhibit low survival rates under hypoxic conditions, limiting their therapeutic efficacy while potentially promoting tumor growth or metastasis [11-12]. These limitations hinder MSC therapeutic applications. In response, researchers are optimizing in vitro culture conditions to enhance MSC biological properties. For example, Li et al. enhanced MSC migration by modifying substrate surface roughness [13], while Liu demonstrated that low pH conditions suppress nucleus pulposus MSC proliferation and promote apoptosis [14]. Recent studies have increasingly focused on identifying optimal in vitro culture conditions to improve MSC quality and functionality, thereby addressing current therapeutic limitations.

This review systematically examines the effects and mechanisms of hypoxic conditions on MSC characteristics from four perspectives: proliferation capacity, migration ability, apoptosis, and differentiation potential. The findings may guide the development of improved in vitro culture environments for MSCs and offer potential strategies to overcome current clinical challenges in MSC-based therapies.

2. Effects of hypoxia on MSC biological characteristics

2.1. Proliferative capacity

Continuous hypoxic culture in vitro has been shown to enhance the proliferative capacity of MSCs, as evidenced by multiple studies. For instance, human umbilical cord Wharton’s jelly-derived MSCs exhibited increased proliferation under 5% oxygen concentration [15]. Guo et al. found that nucleus pulposus-derived MSCs cultured under 2% oxygen entered the doubling phase earlier, with accelerated proliferation rates compared to normoxic conditions. Additionally, hypoxia significantly upregulated the expression of proliferation-related genes CCND1 and MYC in these cells [14]. Since cell proliferation involves a cell cycle that produces two daughter cells, the distribution of cells across cell cycle phases can reflect MSC proliferative activity. Analysis of G0/G1 and S phase cell populations revealed that human umbilical cord MSCs cultured under 3% oxygen (passage 7) exhibited fewer cells in the G0/G1 phase and an increased proportion in the S phase compared to normoxic controls, further confirming hypoxia-induced proliferation enhancement [16].

Although the precise mechanisms underlying hypoxia-mediated proliferation remain unclear, several hypotheses have been proposed. Proliferating Cell Nuclear Antigen (PCNA), a nuclear protein that facilitates DNA polymerase δ activity during DNA synthesis, serves as a marker of proliferative capacity. Xiong et al. compared PCNA expression in rat bone marrow MSCs cultured under 1% O2, 5% O2, and 18% O2, revealing significantly higher PCNA levels in the 1% hypoxia group. This suggests a potential association between hypoxia-induced proliferation and PCNA upregulation, though its mechanistic role requires further exploration [17]. Other studies propose that hypoxia-enhanced proliferation may depend on extracellular signal-regulated kinase (ERK). Hypoxic conditions promote ERK phosphorylation, activating downstream signaling pathways that regulate cell cycle progression and proliferation. Several reports have confirmed elevated phosphorylated ERK levels in hypoxically cultured MSCs [18-20].

However, divergent responses have been observed across MSC tissue sources. For example, Li et al. reported that hypoxia slightly suppressed the proliferation of rat intervertebral disc-derived MSCs, albeit to a limited extent [21]. These findings highlight the tissue-specific variability in MSC responses to hypoxic conditions.

2.2. Migratory ability

The efficacy of MSCs in cell and gene therapy hinges on their homing ability—the capacity to spontaneously migrate to injured or diseased sites for tissue repair. Therefore, identifying optimal in vitro culture conditions to enhance migratory capacity is critical for maximizing MSC therapeutic potential.

Numerous studies have demonstrated that hypoxic culture enhances MSC migration. For instance, Lee et al. reported a significant increase in MSC migratory capacity after 6–18 hours of culture under 2.2% oxygen [22]. Similarly, Transwell migration assays revealed enhanced migratory efficiency in bone marrow-derived MSCs from Sprague-Dawley (SD) rats under hypoxia compared to normoxia [23].

The modulation of migratory capacity is closely linked to the stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 [24-27]. MSCs express CXCR4 on their surface, and its expression is regulated by various factors [28-29]. He et al. demonstrated that 24-hour culture under 2% oxygen significantly upregulated CXCR4 expression in adipose-derived MSCs, with a positive rate of 56.5%, and enhanced SDF-1-directed migration[10]. Hypoxia-induced CXCR4 overexpression correlates with improved migratory ability. Furthermore, supplementing hypoxic cultures with 120 g/mL SDF-1 markedly amplified MSC chemotactic responses under low oxygen, further validating the mechanistic role of hypoxia in modulating migration [23].

2.3. Apoptosis

After setting MSCs48h in the hypoxic group with 1%, 3%, 5% and 10% oxygen, by comparing the number of human amniotic MSCs in the normoxic group, Tang et al found that the percentage of apoptosis in all hypoxic groups was lower than that in the normoxic group, confirming that continuous culture under hypoxic conditions reduced apoptotic [30].

In addition, due to the acute cell damage caused by early hypoxia stress, cells will regain their activity after continuous culture under low oxygen for a period of time, which is also supported by Li et al. Apoptosis was increased at 1% oxygen for 6h and 12h, but at up to 24h and 72h [16]. At the same time, Song also showed that the apoptosis rate of 3% hypoxic group was significantly lower than that of 20% normoxic group, and again confirmed that hypoxic continuous culture could reduce apoptosis [16].

Currently, the mechanism by which hypoxia suppresses apoptosis in MSCs is still being gradually explored. Adams shows that the inhibition of apoptosis in MSCs by hypoxia involves two important oncogenes, Bax and Bcl-2, and apoptosis is regulated by the ratio of the two. Bax is to promote apoptosis, while Bal-2 can inhibit apoptosis [31]. By real-time PCR, proapoptotic Bax mRNA expression in each hypoxic pretreated group was lower than the normoxic control group, while Bal-2 mRNA expression was higher in the hypoxic group than [30] in the normoxic group. Zhu et al also concluded that Bal-2 expression and diminished Bax expression were enhanced in MSCs under persistent hypoxia, which also confirmed the anti-apoptotic effect of persistent hypoxia on MSCs [32]. In addition, the reason for short-term hypoxia and apoptosis is that hypoxia causes the decrease of MMP in MSCs, which then leads to the release of cytochrome C and the activation of cysteine protease-3 (Caspase-3). At the same time, the mitochondrial electron transport chain in cells will also produce a large amount of reactive oxygen species (ROS). The excessive accumulation of ROS will destroy the DNA and protein of cells, which will lead to the apoptosis of cells. However, the duration and intensity of hypoxia treatment can also enhance mitochondrial function and reduce apoptosis [33].

2.4. Multiplication capacity

Mesenchymal stem cells (MSCs), a type of adult stem cell with multilineage differentiation potential, can differentiate into various tissue types and have been actively applied in clinical therapies. Hypoxic culture conditions enhance the differentiation capacity of MSCs. For example, Guo et al. evaluated the osteogenic potential of bone marrow-derived MSCs under physiological hypoxia by measuring osteogenesis-related gene expression (Opn, Alp), protein levels (RUNX2, OPN), and key transcription factors (Runx2). These results demonstrated that all assessed markers were elevated under hypoxia compared to normoxic controls [34]. Additionally, in vivo transplantation of hypoxia-preconditioned human bone marrow-derived MSCs promoted chondrocyte proliferation and cartilage-like tissue formation in germ-free mice [35].

During hypoxic culture, MSCs exhibit distinct differentiation characteristics and mechanisms depending on the target cell lineage [36]. For instance, studies on chondrogenic differentiation revealed that hypoxia promotes cartilage formation by modulating the KDM6A/SOX9 signaling pathway. Hypoxia activates the expression of lysine-specific demethylase 6A (KDM6A), which demethylates the promoter of the chondrogenic transcription factor SOX9, thereby enhancing SOX9 transcription and driving MSC chondrogenesis [37-38]. In contrast, Li et al. inhibited HIF-1α nuclear accumulation and transcriptional activity using the blocker 2-MeOE2, finding no difference in the expression of tenogenic differentiation marker genes (SCX mRNA, TNMD mRNA) between hypoxic and normoxic groups. This indicates hypoxia-specific mechanisms in tenogenic differentiation [39].

However, the effects of hypoxia on MSC differentiation are not universally consistent. Cicioned et al. reported reduced adipogenic and osteogenic differentiation capacities in human bone marrow-derived MSCs after 21 days of induction under 1% oxygen [40]. Conversely, Martin-Rendon et al. observed no significant differences in differentiation outcomes between hypoxic (1.5% oxygen) and normoxic cultures after the same duration [41]. These discrepancies suggest that hypoxia-mediated effects on MSC differentiation may depend on oxygen tension levels, tissue sources, exposure duration, and other variables, leaving the debate over its pro-differentiative or inhibitory roles unresolved.

3. Discussion

Hypoxic environments can enhance the proliferation capacity, migration potential, differentiation capability, and inhibit apoptosis of mesenchymal stem cells (MSCs). However, due to variations in tissue sources and other culture conditions, conflicting experimental results persist. The precise mechanisms underlying hypoxia’s effects on these characteristics remain unclear, necessitating further investigation into this topic.

Other studies have explored additional properties of MSCs under hypoxia. Guo et al. demonstrated improved stemness maintenance in hypoxic conditions by detecting elevated expression levels of pluripotency-related genes OCT4, NANOG and SOX2[16]. Another study revealed enhanced adhesion capacity in bone marrow-derived MSCs after 24 hours of culture under 2% oxygen [42]. Hypoxia also affects mitochondrial dynamics in MSCs, inducing denser and thicker tubular mitochondrial morphology. Compared to normoxic conditions, hypoxia regulates mitochondrial quality and quantity to sustain normal metabolic activities in MSCs [43-44]. Collectively, hypoxia has been found to enhance multiple biological properties of MSCs to varying degrees.

Investigating hypoxia’s influence on MSC performance is essential for determining the optimal oxygen concentration for in vitro culture, thereby optimizing their efficacy and improving their survival and functionality within recipients. Despite significant advancements in recent years, challenges remain. First, heterogeneity in MSC quality derived from diverse individuals and tissues, coupled with a lack of standardized criteria, may lead to divergent or contradictory results. Second, as the optimal culture conditions remain under investigation and techniques are not yet fully matured, challenges such as difficult isolation or limited accessibility of certain MSCs may hinder experimental efficiency.

As a promising therapeutic approach for various clinical diseases, MSCs cultured under hypoxia-based on this review of hypoxia-induced changes in their properties-may exhibit enhanced therapeutic potential. Establishing optimal culture protocols could enable MSCs to better exert their biological functions in vivo, thereby improving treatment outcomes and expanding their prospects in cell-based therapies.

4. Conclusion

Despite significant advancements in current research on mesenchymal stem cells (MSCs), numerous challenges persist. This paper focuses on the effects of hypoxic conditions on MSC characteristics-specifically proliferation, migration, apoptosis, and differentiation-and explores related mechanisms. By comparing hypoxic-treated MSCs with normoxic groups, based on reviewed literature, the findings of this paper demonstrate that hypoxia enhances MSC proliferation, migration, and differentiation capacities while suppressing apoptosis. Further analysis of existing experimental methodologies and conclusions reveals potential mechanisms underlying these effects. For instance, improved proliferation under hypoxia correlates with elevated PCNA expression and ERK phosphorylation, while enhanced migration is primarily mediated by the SDF-1/CXCR4 axis.

However, certain limitations exist in this review. Due to incomplete literature coverage, the analysis of hypoxia-induced changes in MSC properties remains insufficiently comprehensive. Current evidence suggests that hypoxia’s impact on MSCs involves multifactorial constraints, leaving precise outcomes and detailed mechanisms unresolved. Future research should focus on advancing mechanistic understanding and keeping pace with emerging developments in the field to facilitate the clinical translation of MSCs and maximize their therapeutic potential.