Application of autophagy in mesenchymal stem cells

Abstract

In this editorial, we have taken an in-depth look at the article published by Wan et al. The study showed that preconditioning mesenchymal stem cells (MSCs) protected them against programmed cell death, and increased their survival rate and therapeutic potential. Autophagy, a type of programmed cell death, is a major intracellular degradation and recycling pathway that is crucial for maintaining cellular homeostasis, self-renewal, and pluripotency. We have explored the relationship between autophagy and MSCs to determine the role of autophagy in the therapeutic applications of MSCs.

Key Words: Autophagy; Mesenchymal stem cells; Programmed cell death; Apoptosis; Bone marrow mesenchymal stem cells; Umbilical cord mesenchymal stem cells

Core Tip: Reducing the programmed death of pre-conditioned bone marrow-derived mesenchymal stem cells can significantly enhance their engraftment, survival, and differentiation potential, and modulate the immune microenvironment of the recipient tissue. Autophagy, an important complementary pathway of programmed cell death, plays a crucial role in cellular homeostasis, self-renewal, and functional regulation. Regulating autophagy in mesenchymal stem cells provides a new perspective and strategy for stem cell therapy.

INTRODUCTION

Programmed cell death (PCD), a genetically controlled mode of active cell death, plays a crucial role in the development and homeostatic maintenance of organisms[1]. Autophagy is an important complementary pathway to PCD, and has gained attention in recent years for its role in cellular homeostasis, self-renewal, and functional regulation. The mesenchymal stem cells (MSCs) are known to attenuate neuronal damage and dysfunction by regulating autophagy, thus improving the prognosis of neurodegenerative diseases[2]. Furthermore, MSCs and their exosomes can alleviate hepatic injury by promoting the autophagic flux in liver cells[3]. Autophagy may also mediate the therapeutic effects of MSCs in cardiovascular diseases, diabetes, and autoimmune diseases[4]. Taken together, MSCs can improve the microenvironment of damaged tissues and promote tissue repair and regeneration by regulating autophagy.

As studies increasingly delve into the functional link between autophagy and MSCs, and discover new mechanisms and targets of autophagy in MSCs, we can expect more effective strategies for the clinical application of MSCs. At the same time, the complexity and diversity of autophagy regulation in the MSCs need to be elucidated to avoid potential adverse effects (Figure 1).

Figure 1 This image introduces the logical framework of this article. This image consists mainly of including these parts: (1) Programmed cell death; (2) Applications of autophagy; (3) Regulation of autophagy in mesenchymal stem cells; (4) Two common types of autophagy; (5) Methods for pretreatment of mesenchymal stem cells; and (6) Pretreatment of mesenchymal stem cells in autophagy. HIF-1α: Hypoxia-inducible factor-1α; mTOR: Mammalian target of rapamycin; MSCs: Mesenchymal stem cells; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β.

PCD

PCD is a genetically controlled, active form of cell death as opposed to necrosis, which is a passive process caused by external physical, chemical or biological factors. Necrosis is often accompanied by the rupture of cell membranes and the release of cellular contents, which can trigger an inflammatory response. In contrast, the cell membrane remains intact during PCD, no inflammatory response is elicited, and the entire death process requires energy expenditure. Various types of PCD have been discovered so far, including apoptosis, necroptosis, pyroptosis, autophagy, ferroptosis and cuproptosis.

Apoptosis, the most prevalent and extensively researched type of PCD, is characterized by chromatin condensation and margination, cellular shrinkage, phosphatidylserine externalization on the inner leaflet of the cell membrane, and the formation of apoptotic bodies through cellular exocytosis[5]. It is triggered by multiple stimuli, and is essential during embryonic development and aging to maintain cellular homeostasis. In addition, apoptosis also plays a pivotal role in defending against autoimmune responses and in the elimination of damaged cells[6]. The apoptotic cascade is driven by the endogenous (mitochondrial) and the exogenous (death receptor-mediated) pathways, which are primarily controlled by members of the Bcl-2 family[7] and caspase family[8] of genes. Dysregulated apoptosis is a key contributor to the onset and progression of cancer. Furthermore, neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease, as well as autoimmune disorders, are closely linked to aberrant apoptosis. The study titled “Pretreatment can alleviate programmed cell death in mesenchymal stem cells” which was published by Wan et al[9] has shown that preconditioning MSCs with prostaglandin E1 upregulates hypoxia-inducible factor-1α (HIF-1α)[10]. This finding offers significant insights into the therapeutic advantages of MSCs in combating pulmonary hypertension. Necroptosis is a mode of PCD that is triggered by death receptor signaling via the receptor interacting protein kinase 1-receptor interacting protein kinase 3-mixed lineage kinase domain-like pathway[11]. Unlike apoptosis, necroptosis culminates in the rupture of cell membrane, resulting in the discharge of intracellular factors that can initiate an inflammatory reaction.

Pyroptosis, also known as cellular inflammatory necrosis, is a newly discovered mode of PCD. It is triggered by activation of the inflammasome complex, and manifests as continuous swelling of the cell until the plasma membrane ruptures and releases pro-inflammatory cellular contents. Six primary inflammasomes have been identified so far: Nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3, nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 1, NLR family CARD domain-containing protein 7, ice protease-activating factor, NLR family CARD domain-containing protein 4, and absent in melanoma 2[12]. During pyroptosis, several members of the caspase family are activated, which cleave the pore-forming gasdermin family proteins[13]. The gasdermins then perforate the cell membrane, thereby facilitating the release of inflammatory mediators. Pyroptosis is pivotal in the pathology of various diseases, including cancer[14], inflammatory disorders, and neurodegenerative diseases. Modulating the initiation and progression of pyroptosis could potentially mitigate these conditions.

Ferroptosis represents a unique form of iron-dependent, regulated cell death, which is initiated by the buildup of lipid peroxides in cellular membranes[15]. Iron ions facilitate peroxidation of polyunsaturated fatty acid phospholipids through the Fenton reaction, resulting in cellular membrane damage and subsequent cell death. This process is tightly regulated by intracellular antioxidant systems, particularly glutathione peroxidase 4, which is able to reduce phospholipid peroxides and inhibit iron-mediated death[16]. The induction and inhibition of ferroptosis in MSCs has emerged as a potential therapeutic strategy for anti-tumor treatment.

Cuproptosis is a copper-mediated form of regulatory cell death that is caused by the inhibition of the tricarboxylic acid cycle, proteotoxic stress and mitochondrial dysfunction[17]. It is regulated by various signaling pathways, including those involved in the generation of reactive oxygen species (ROS) and the breakdown of solute carrier family 7 member 11[18]. Copper chelators, such as D-penicillamine and tetrathiomolybdate ammonium[19], can potentially inhibit the growth of cancer cells by modulating cuproptosis.

THE ROLE OF AUTOPHAGY IN CELLULAR HOMEOSTASIS

Autophagy is a self-catabolic process wherein the cytoplasmic contents or organelles are enveloped by a bilayer membrane into an autophagosome[20], which subsequently fuses with lysosomes to form an autolysosome, resulting in the degradation of the encapsulated contents[21]. While autophagy is essential for recycling cellular components and maintaining homeostasis, excessive autophagy can lead to type II cell death[22]. Based on the mechanism of action, autophagy can be categorized into three types - macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy is the most common type, and involves the encapsulation and degradation of macromolecules and organelles in the autophagosomes[23]. In contrast, microautophagy does not require formation of autophagosomes as the lysosomes or vacuoles directly engulf and degrade substances in the cytoplasm[24]. During chaperone-mediated autophagy, molecular chaperones such as HSC70 selectively recognize and bind to soluble proteins in the cytoplasm, and transport them to lysosomes for degradation[25].

Autophagy is triggered in response to starvation, hypoxia, endoplasmic reticulum stress, and other stressors. These signals lead to the activation of autophagy-related genes (Atg) via the mammalian target of rapamycin (mTOR)[26] pathway. The Atg-mediated formation of autophagosomes is orchestrated by multiple autophagy-related proteins, including but not limited to Beclin-1[27], ATG14, and VPS34. The autophagosome membrane continuously extends, enveloping large molecules and organelles in the cytoplasm, ultimately self-closing to form mature autophagosomes. The fusion of autophagosomes with lysosomes results in autolysosomes, wherein the cellular contents are digested by the lysosomal enzymes. The degradation products, including amino acids and fatty acids, are then released back into the cytoplasm for cellular recycling[21]. Autophagy not only breaks down intracellular macromolecules to provide energy and raw materials for the cell during starvation or nutritional deficiency, but also contributes to cell renewal and repair by degrading aberrant or aging organelles. In addition, autophagy can remove intracellular pathogens like bacteria and viruses, as well as abnormal protein aggregates, thereby safeguarding cells against infection and damage[28].

Autophagy and apoptosis exhibit a synergistic relationship in the event of cellular damage or stress. In fact, autophagy can serve as an upstream regulator of apoptosis by breaking down damaged organelles or proteins, thus supplying the essential signals or raw materials for the apoptotic process. For instance, tumor necrosis factor-α (TNF-α) and other signaling molecules that are upregulated during autophagy may initiate the apoptotic cascade by activating critical proteins involved in the pathways[29]. Furthermore, excessive accumulation of autophagosomes or a saturation in their degradation capacity can also induce a shift in the cell death pathway. However, the metabolic regulation of autophagy is often at odds with apoptosis. For example, under conditions of starvation or oxidative stress, autophagy can inhibit apoptosis by reducing the abundance of pro-apoptotic proteins in the cytoplasm.

AUTOPHAGY IN MSCs

MSCs are adult stem cells that primarily originate from the mesoderm, and exhibit self-renewal and multipotent differentiation abilities. The MSCs have been detected in various fetal and adult tissues, including but not limited to bone marrow, placenta, umbilical cord, adipose tissue, mucous membranes, bones, muscles, lungs, liver, pancreas, and even amniotic fluid and umbilical cord blood[30]. Under suitable in vivo or in vitro conditions, MSCs can differentiate into various cell types, such as osteoblasts, chondrocytes, adipocytes, myoblasts, neuronal cells, hepatocytes, endothelial cells, etc. Depending on their source, MSCs are broadly classified as bone marrow MSCs (BM-MSCs), umbilical cord MSCs (UC-MSCs), adipose MSCs, and amniotic membrane MSCs[31].

The complexity and diversity of autophagy regulation in MSCs

Both intrinsic and extrinsic factors affect autophagy in the MSCs. Intrinsic factors include cell signaling pathways and mitochondrial function, and the extrinsic factors include nutrients, hypoxia, extracellular matrix, drugs, and chemicals. Multiple signaling pathways are involved in the regulation of autophagy in MSCs. For example, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR signaling pathway is a major negative regulatory pathway of autophagy. Hair follicle MSC-derived exosomes inhibit the PI3K/AKT/mTOR signaling pathway via miR-214-3p, which in turn maintains mitochondrial dynamic stability and enhances mitochondrial autophagy[32]. Furthermore, the AMP-activated protein kinase (AMPK) signaling pathway can activate autophagy by inhibiting the mechanistic target of rapamycin complex 1 activity[33]. ROS produced by mitochondria act as signaling molecules to activate autophagy in order to scavenge damaged mitochondria and maintain intracellular redox homeostasis[34]. In addition, changes in the membrane potential of mitochondria can affect autophagy[35].

MSCs acquire energy and nutrients during starvation by activating autophagy. For example, glucose deficiency induces the onset of autophagy in MSCs, whereas glucose supplementation inhibits autophagy[36]. Furthermore, MSCs can adapt to hypoxic conditions by activating autophagy, and maintain survival and function. HIF-1α plays an important role in hypoxia-induced autophagy in MSCs[37]. Components of the extracellular matrix, such as fibrinogen, can influence autophagy in MSCs by activating cell surface receptors, which in turn regulate intracellular signaling pathways[38]. Several drugs and chemicals are known to modulate autophagy in MSCs. For example, rapamycin is an mTOR inhibitor that inhibits autophagy by activating mTOR activity[39]. In addition, natural compounds such as resveratrol[40] and curcumin[41] have been shown to regulate autophagy in MSCs.

Two typical types of MSCs

The UC-MSCs are most promising for clinical application on account of their high quality, purity, and quantity, as well as the convenience of collection and lack of ethical concerns[42]. Nevertheless, as MSCs were first discovered in the bone marrow, BM-MSCs are the more widely used in cell therapy and tissue engineering[43]. Recent studies suggest that autophagy may play a key role in the biology of BM-MSCs and UC-MSCs.

BM-MSCs: BM-MSCs mainly exist in the bone marrow cavity, and in connective tissues such as trabecular bone, skeletal muscle, and periosteum. Autophagy plays a role in the proliferation, differentiation, and therapeutic potential of BM-MSCs. Pierrefite-Carle et al[44] hypothesized that autophagy provides BM-MSCs with energy substrates during their differentiation. The authors observed significant accumulation of autophagic vacuoles in the undifferentiated BM-MSCs, along with stagnant autophagic flow. In addition, the autophagosome marker LC 3-II was downregulated in the BM-MSCs during the early stages of osteogenic differentiation (within 12 hours of culture), suggesting that these autophagic vacuoles may serve as a source of energy substrates for the differentiation process. The significance of autophagy in BM-MSCs is further underscored by its role in the progression of bone degenerative diseases, such as disc degeneration and osteoarthritis, as well as in bone metabolic disorders like osteoporosis, metaplastic osteitis, and osteosclerosis. Autophagy modulators, such as mechanically targeted rapamycin kinase inhibitors, AMPK activators, and phytochemicals, have been shown to promote bone regeneration[45]. Wu et al[46] showed that hypoxic conditions induced autophagy in the BM-MSCs in vitro through the activation of the extracellular signal-regulated kinase-1/2 pathway. Furthermore, Zheng et al[47] found that senescent BM-MSCs have enhanced autophagic activity as well as increased levels of inflammatory factors such as interleukin (IL)-6 and IL-8, which upregulate intracellular FoxO3a protein levels. Modulating autophagy and FoxO3a expression could delay BM-MSC senescence and improve its therapeutic efficacy. Li and Qu[48] showed that the PI3K/Akt/nuclear factor-kappaB, mitogen-activated protein kinase/extracellular signal-regulated kinase, and stem cell factor/c-kit pathways are involved in the interaction between autophagy and apoptosis, and that modulation of autophagic activity could potentially improve the therapeutic efficacy of BM-MSCs against myocardial infarction.

UC-MSCs: UC-MSCs are versatile stem cells that exist in the newborn’s umbilical cord, predominantly within Wharton’s Jelly[49], and can differentiate into bone, cartilage, fat, muscle, tendon, nerve, liver, cardiac muscle, and other cell types in vitro in response to suitable induction factors. UC-MSCs can express surface markers such as CD105, CD73, and CD90[50].

UC-MSCs can reduce immune rejection to allografts by suppressing the activation of immune cells. Additionally, they can facilitate hematopoietic recovery[51] and enhance the engraftment of hematopoietic stem cells. The biological function and therapeutic efficacy of the UC-MSCs can be enhanced by regulating autophagic activity. Moreover, UC-MSCs themselves can be used as autophagy modulators. For example, in inflammatory diseases, the transplantation of UC-MSCs can inhibit the production and release of inflammatory factors, thereby reducing inflammatory responses and tissue damage.

He et al[52] showed that autophagy is crucial for the function of UC-MSCs-derived exosomes (UC-MDEs), and detected high expression of the autophagy markers BECN1 and MAP 1 LC3B in UC-MDEs by transmission electron microscopy. Furthermore, the autophagy inhibitor 3-methyladenine significantly reduced the ameliorative effect of UC-MDEs on glycolipid metabolism in type 2 diabetic rats. In a study conducted by Ma et al[53] exosomes from TNF-α-preconditioned UC-MSCs inhibited autophagy in the acinar cells of severe acute pancreatitis by shuttling 3,4-dihydroxyphenylglycol and suppressing the mTOR pathway, and alleviated the symptoms of severe acute pancreatitis. Furthermore, Han et al[54] showed that UC-MSCs can promote diabetic wound healing by inducing autophagy, which may have potential clinical applications. Wang et al[55] showed that 100 nM to 10 μM rapamycin induced autophagy in UC-MSCs, and the optimal dose was 100 nM. Enhancing autophagy in the UC-MSCs improved the pro-angiogenic activity of the conditioned medium, which in turn promoted wound healing and tissue repair. Ma et al[56] showed that the combination of UC-MDEs and autophagy activators significantly enhanced the function of human corneal epithelial cells and attenuated corneal defects, apoptosis, and inflammation through the activation of the AMPK/mTOR/ULK1 pathway, thereby providing a new therapeutic strategy for corneal wound healing and ocular surface regeneration. Yin et al[57] were able to restore ovarian function in mice and increase the circulation of CD8⁺CD28^- T cells using UC-MSCs. Mechanistically, the heme oxygenase-1 expressed in these UC-MSCs induced autophagy in the ovarian cells by activating the JNK/Bcl-2 signaling pathway. Overall, these studies show that modulation of autophagic activity in MSCs promotes self-renewal, enhances multipotent differentiation, delays aging, and improves immunomodulation.

Methods and effects of MSCs by pretreatment

Physical preconditioning factors include hypoxia, mechanical stimulation, and electromagnetic radiation. Hypoxic preconditioning can mimic the in vivo microenvironment and upregulate HIF-1α, which in turn promotes angiogenesis and metabolism in the MSCs by transcriptionally activating the downstream genes[58]. Mechanical stimulation, such as low intensity focused pulsed ultrasound, can effectively stimulate human UC-MSCs in vitro, reduce thyroid cell apoptosis, improve thyroid function, and reduce excessive accumulation of autoimmune antibodies in vivo[59]. Electromagnetic radiation generally includes low-intensity laser exposure and magnetic field stimulation. Magnetic field stimulation can affect cell membrane potential and ion channel activity in MSCs, which in turn regulates metabolism and cellular function[60].

Chemical pre-treatment includes drugs, active oxygen, etc. Some drugs can modulate intracellular signaling pathways and gene expression in the MSCs. For example, rapamycin pretreatment inhibits mTOR activity and upregulates autophagy in MSCs, thereby improving their survival under stressful conditions[39]. In addition, 5-azacytidine promotes the differentiation of MSCs to specific cell types[61]. Appropriate amounts of ROS activate intracellular stress responses and antioxidant defense mechanisms. ROS pretreatment can enhance the survival of MSCs and increase their resistance to apoptosis by modulating the antioxidant enzyme system and intracellular signaling pathways[62].

Cytokines play an important role in the proliferation, differentiation, and immunomodulation of MSCs. Inflammatory cytokines, such as TNF-α[63] and IL-1β[64], can activate the immunoregulatory and anti-inflammatory functions of MSCs. Simultaneous or sequential use of two or more pretreatment methods can synergistically enhance the therapeutic effects of MSCs. For example, drug preconditioning combined with mechanical stimulation protected MSCs against apoptosis and enhanced their function[65].

Relationship between preconditioned MSCs and autophagy

Pretreatment of MSCs maintains cellular homeostasis and survival, and regulates immune function through autophagy. Therefore, pretreated MSCs exhibit enhanced viability and differentiation abilities, which translate to improved therapeutic efficacy in tissue repair and regeneration. For example, Yang et al[66] established TNF α-licensed exosome-immobilized titanium surfaces to correct macrophage immune status and accelerate osseointegration in type 2 diabetic conditions by activating autophagy. Furthermore, pretreatment can also enhance the immunomodulatory and anti-inflammatory functions of MSCs. Pretreatment of MSCs by modulating the level of autophagy offers a novel treatment strategy for various diseases. For example, selenomethionine promoted the production of MSC-derived extracellular vesicles and increased the delivery of miR-125a-5p in MSC-derived extracellular vesicles, which enhanced the protective effects of MSC-derived extracellular vesicles on attenuating nucleus pulposus cellular senescence and mitigating disc degeneration[67].

CONCLUSION

Given their unique biological properties and wide range of applications, MSCs occupy a pivotal position in the field of medical research. Our study shows that autophagy plays a crucial role in MSCs, and interfering with the autophagic process, either through targeted drugs or other intervention strategies, can optimize their therapeutic effects. In the recent study “Pretreatment can alleviate programmed cell death in mesenchymal stem cells” published by Wan et al[9] in World Journal of Stem Cells, the researchers showed that reducing programmed death of MSCs by preconditioning can significantly enhance their engraftment, survival and differentiation potential, and modulate the immune microenvironment.