A Review in Regenerative Medicine: Human Umbilical Cord Mesenchymal Stem Cells in Activating the PI3K/AKT/mTOR Pathway

Introduction

Human umbilical cord mesenchymal stem cells (hUC-MSC) have been a recent area of interest within regenerative medicine [2]. hUC-MSCs require less invasive harvesting methods than other stem cells and are more concentrated [2]. They are mainly harvested from the area of the umbilical cord called Wharton’s Jelly, where fragments are isolated and cultured for experimental use [6]. The opening of the first stem cell bank in 2004 has allowed for umbilical cord stem cells to be applied in current research instead of discarded [8]. hUC-MSCs are currently being studied to treat a variety of illnesses involving tissue regeneration, including premature ovarian failure (POF) and osteoporosis. These illnesses affect millions of people globally each year, but current research is limited to pre-clinical phases as researchers try to further elucidate the underlying mechanisms for effective treatment [2]. Despite their different circumstances, both of these conditions have currently responded to in vivo treatments with hUC-MSCs. In cases with POF, researchers are looking to regenerate the follicles needed to regain ovarian function [2]. Similar effects can be viewed in osteoporosis treatments with hUC-MSC transplantation through the recuperation of osteocytes [6].

In association with hUC-MSC, the phosphoinositide 3-kinase/protein kinase B/mammalian target of the rapamycin pathway (PI3K/AKT/mTOR) is a secretory pathway responsible for regulating a variety of cell functions [1]. The PI3K/AKT/mTOR pathway is an intracellular signal pathway found within eukaryotic cells, and researchers have found that the PI3K/AKT/mTOR pathway is often triggered by the paracrine effect of hUC-MSCs [1]. hUC-MSCs secrete cytokines such as the Human Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGF), Stem Cell Factor, and Epidermal Growth Factor (EGF) [3,8]. These cytokines trigger the cascade effect of the PI3K/AKT/mTOR pathway by attaching to the receptors in the plasma membrane. The collaboration between cytokines and the pathway also aids in regulating the effects of cell proliferation, as the growth of new cells is an essential target for treatment.

Essential to cell multiplication, proliferation regenerates new cells and increases tissue growth, aiding in recovering damaged cells affected by overactive cell processes. Researchers have found that hUC-MSCs contain high proliferation abilities that can be applied towards current research [2]. Notably, hUC-MSCs can reduce autophagy and apoptosis–both balancing factors that keep systems in the body functioning properly. Autophagy is a process that recycles unwanted organelles and other unnecessary factors, maintaining balance within the cell [2]. Meanwhile, apoptosis removes damaged cells that could potentially cause harm. Reducing the self-digestion caused by autophagy can increase the amount of mature oocytes present, improving ovarian function. A better understanding of the mechanisms regulating these processes can lead to advancements in treating conditions that millions of people suffer from each year. Researchers have found that these processes can often be regulated through the cascade effect of the PI3K/AKT/mTOR pathway [4].

The balance between regulating proliferation or promoting cell differentiation often determines the use of the hUC-MSCs in current research. The processes between the hUC-MSCs and the PI3K/AKT/mTOR are commonly interdependent. However, due to their novelty, most of the mechanisms in this relationship are still unknown. Therefore, researchers often observe associated disorders–like POF or osteoporosis–to further their understanding and use hUC-MSCs more effectively. It is important to note that excessive proliferation due to increased activation of the PI3K/AKT/mTOR can have adverse effects on the patient's health since uncontrollable proliferation results in tumors–otherwise known as tumorigenicity. This review will discuss the potential of using hUC-MSCs in conjunction with the PI3K/AKT/mTOR for future regenerative treatment. It will also evaluate potential alternatives in current treatment to limit the effects of tumorigenicity and excessive proliferation of hUC-MSCs.

The Use of hUC-MSCs in Current Treatment

Premature Ovarian Failure

Premature Ovarian Failure (POF) is one of the leading causes of infertility in women. Approximately 1% of women under 40 are currently affected worldwide, with evidence of a recent increase within this demographic [2]. At birth, females are born with a fixed number of oocytes determined by the primordial follicles. Oftentimes with age, the quality of the follicles decreases and female fertility dwindles [2]. hUC-MSCs have been recently identified to treat this decrease in female fecundity by promoting primordial follicle growth [3]. Researchers aim to increase the amount of viable follicles to promote ovarian function and enhance fertility in females struggling with infertility.

In a recent mice study, researchers targeted Granulosa Cells (GCs) as a possible treatment option for POF [2]. GCs are linked to follicular development through estradiol secretion, aiding in the maturation of oocytes [2]. This experiment contained a control group of POF afflicted mice and an experimental group of POF mice treated with hUC-MSCs. The experimental group was treated with hUC-MSCs via tail vein injections at 14 days since hUC-MSCs have antioxidative properties that help decrease the autophagy levels of GCs [2]. Decreasing the autophagy levels will reduce the stress on follicle production, increasing fertility [1, 2]. This study also measured the levels of different ovarian hormones, such as anti-Mullerian hormone (AMH), follicle stimulating hormone (FSH), estradiol (E2), and the luteinizing hormone (LH) on POF mice treated with hUC-MSCs. In measuring these indicator hormones for ovarian function in reproduction, the effect of hUC-MSCs on fertility was observed. The results determined that the hormone levels changed substantially when POF mice were treated with hUC-MSCs. Compared to the control mice with POF, those treated with hUC-MSCs saw an increase in AMH and E2, and a decrease in both FSH and LH, which work alongside each other. Changing FSH and LH levels aid in follicle development since they help regulate the estrogen secretion of GCs [2].

The secretion of Vascular Endothelial Growth Factor (VEGF) notably increased during treatment [2]. VEGF has the capacity to decrease LC3B expression, a key protein involved in autophagy expression. Immunofluorescence determined the decreased expression of LC3B in the POF+hUC-MSC treatment compared to the POF treatment [2]. Additionally, the secretion of VEGF activates the PI3K/AKT/mTOR pathway and mediates the regulation between the expression of these cell factors and the folliculogenesis that increases fertility. Tracking the levels of proteins and hormones during treatment allows researchers to correlate the mechanisms between the hUC-MSC paracrine effect and how it activates the PI3K/AKT/mTOR pathway. Without this correlation, researchers would be unable to determine whether hUC-MSCs had any real effect on the treatment.

On another note, recent concerns have emerged regarding the viability of hUC-MSCs after transplantation [3]. A study conducted in 2022 suggests the use of hyaluronic acid (HA) to increase the viability of the stem cells [3]. HA is naturally occurring within the extracellular matrix and its ability to increase paracrine function proves crucial to treatment processes. Researchers were specifically interested in paracrine function because they believed these paracrine mechanisms and cell-to-cell interactions were just as notable as differentiation in ameliorating the effects of POF [3]. Since much of the research concerning hUC-MSCs is still in the pre-clinical phase, exploring the effects of HA can be beneficial when extending cell longevity during experiments. Compared to the previous experiment where VEGF was critical in activating the regulation of the PI3K/AKT/mTOR pathway, the study conducted by Jiao et al. viewed Hepatocyte growth factor (HGF) as a key component in follicle survival [2, 3]. When secreted by hUC-MSCs, HGF has antioxidative mechanisms that are capable of horizontally transferring genes to the cell–a mechanism that allows for cells to share information without being directly related [1]. Researchers also observed whether HA will have any effect on the genetic composition of cells. Of the 29,207 genes viewed in the HA treatment, 99.99% of the genes remained the same [3]. Therefore, HA not only increased cell viability, it also did not result in gene mutation within the hUC-MSCs, which was an initial concern for researchers. Both studies were successful in achieving fertility, promoting further exploration of the effects of HA with hUC-MSCs to investigate the actions needed to progress the current research.

Osteoporosis

About 200 million people worldwide live with osteoporosis each year [6]. Those most at risk are elderly and postmenopausal women due to decreased estrogen levels. The balance of bone metabolism for homeostasis relies on the quantity of osteoclasts, osteoblasts, and osteocytes [9]. Researchers believe that the PI3K/AKT/mTOR pathway is responsible for the regulation of osteoblastic proliferation and apoptosis [4]. hUC-MSCs aid in recovering homeostatic regulation through the proliferation of osteoblasts and reabsorption of osteoclasts by the body [6]. These cells work in tandem since osteoclast removes damaged bone cells and osteoblast helps promote new bone growth [6]. Researchers aim to study the potential of hUC-MSC in osteoporosis treatments to regain this homeostasis through its ability to activate the PI3K/AKT/mTOR pathway.

A recent osteoporosis study identified Metformin as a potential drug to use in coculture with hUC-MSCs [6]. Metformin is currently used to treat type 2 diabetes, but researchers are observing its anti-inflammatory and tissue-repairing characteristics to treat osteoporosis [6]. Researchers observed the effect of Metformin on innate immune cells called macrophages in treatment with hUC-MSCs. Notably, M2 macrophages have been found to promote cell proliferation and tissue repair, while M1 macrophages have been observed to encourage the opposite. Macrophages control inflammation cascades as well as immune responses and can be used in tissue repair since macrophages can polarize into different functional states–according to researchers they can polarize to inhibit inflammation or remodel bone [6]. Researchers tested M1 and M2 macrophages treated with Metformin, and observed that the expression of osteogenic proteins was increased in a coculture between M2 and MSCs when the cells were polarized [6]. Since these proteins are linked to bone formation, their increased expression is viewed as a positive correlation between hUC-MSCs and current treatment. Researchers then looked at the PI3K/AKT/mTOR pathway, studying its link between inflammation and bone formation [6]. The transition between inflammation to regeneration for this pathway is attributable to Metformin altering the state of M1 and M2 macrophages, activating the pathway and ultimately increasing osteogenic protein expression. Altering M1 macrophages to M2 macrophages facilitates the desired transition from inflammation to tissue regeneration [6]. The use of Metformin was successful in this study since it stabilized the coculture between M2 macrophages and hUC-MSCs, while osteoblastic differentiation was still regulated by the PI3K/AKT/mTOR pathway [6]. Researchers can further investigate the three main communication pathways between osteoblasts and osteoclasts, which constitutes the direct contact pathway, the paracrine pathway, and the growth factor deposition pathway to view other mechanisms or possibilities for treating osteoporosis with hUC-MSCs.

Limitations with Excessive Proliferation in hUC-MSC Therapy

Researchers must also consider the adverse effects of using hUC-MSCs to increase proliferation when activating the PI3K/AKT/mTOR pathway. A current concern with hUC-MSC transplantation is the chance of tumorigenicity [4,7]. Though this correlation has not been solidified due to the novel aspect of hUC-MSC transplantation, researchers are concerned about the likelihood of cancer when increasing proliferation [5]. Researchers are viewing potential alternatives to overcome this limitation through hUC-MSC derived extracellular vesicles and exosomes. Exosomes are essential to the cell in transporting important molecules like lipids, nucleic acids, proteins, and they participate in paracrine secretion [5]. They carry similar functions as hUC-MSCs without the same limitations as direct transplantation. In using MSC-derived exosomes, it would carry the same function of paracrine secretion as hUC-MSCs, which is needed to trigger the PI3K/AKT/mTOR pathway. However, this would be a cell-free therapy, therefore removing the risk of uncontrollable cell proliferation that can lead to cancer [5].

Direct hUC-MSC Transplantation Alternatives

Narrowing the specific components of hUC-MSCs used also further elucidates their mechanism with the PI3K/AKT/mTOR pathway. Extracellular vesicles (EVs) are small organelles that transfer mRNA, microRNA(miRNA), and other proteins to their target cells [4]. The genetic information carried by EVs is essential for protein synthesis and ensuring proper cell function. EVs are also important for communication between cells. They offer antioxidative, anti-inflammatory, and antiapoptotic effects due to the contents they carry, which are factors that researchers have previously observed in hUC-MSCs to reduce autophagy and increase proliferation [2-4]. Deriving EVs and exosomes from hUC-MSCs can be a key component to prevent the aforementioned limitations in the clinical study.

In a study conducted in 2023, PI3K inhibitor LY294002 was utilized to view the effect of EVs on inducing osteoblasts. After this inhibition, protein expression induced by the EVs did not increase, meaning the osteoblastic induction was also hindered [4]. This result signifies that osteoblastic induction was reliant on the PI3K/AKT/mTOR pathway since the inhibition of the PI3K would not allow for the rest of the pathway to be activated. Utilizing methods like these supports the correlation between hUC-MSCs and the PI3K/AKT/mTOR pathway, portraying the necessity for these stem cells in treatment. Comparatively, a study conducted by Qu et al. demonstrated how miR-126-3p hUC-MSC-exosomes could be used to activate the PI3K/AKT/mTOR pathway [5]. Exosomes were able to transport a known positive regulator for endothelial cell proliferation called miR-126-3p to the target gene PIK3R2. Since PIK3R2 is responsible for creating the PI3K enzyme, increased proliferation of endothelial cells was observed [5]. Researchers are interested in utilizing this relationship as a potential therapeutic target, since miR-126-3p was seen to have pro-angiogenic and anti-apoptotic effects. Exosomes containing miR-126-3p could be potentially utilized to treat POF, as the epithelial cells were seen to increase ovarian function due to miRNA’s decreased apoptosis of ovarian granulosa cells (OGC) [5]. Reducing cell death of OGCs via miR-126-3p allowed for differentiation of endothelial cells that promotes ovarian function, as opposed to hindering it. Further evaluation and research on paracrine secretion of hUC-MSCs is needed to translate these findings into clinical studies. This current alternative to direct hUC-MSC transplantation can work on small scale experiments, but more research needs to be conducted to expand their capabilities.

Conclusion

The use of hUC-MSCs to activate the PI3K/AKT/mTOR pathway exhibits promise in future treatments for regenerative medicine. Current successful preclinical treatments with hUC-MSCs for POF and osteoporosis demonstrate the necessity for further research. The findings from this research have the potential to be expanded to other afflictions as well, reducing the daily struggle of millions globally. The paracrine effect of hUC-MSCs to secrete endocrine factors warrants further exploration in order to overcome excessive proliferation elements that come with stem cell transplantation. On their own, hUC-MSCs are key to the field of regenerative medicine due to their proliferation abilities. However future researchers should focus on maintaining cell viability and preventing excessive proliferation after activating the PI3K/AKT/mTOR pathway.

About the Author: Yadira Yepiz-Yepez

Yadira Yepiz-Yepez is a fourth year Cell Biology major at UC Davis. Her excitement surrounding cells began in her high school biology class when she watched a documentary on gene cloning which introduced her to the world of scientific research. Since then, she has found a passion for stem cell research and hopes to pursue this facet of biology after graduation. As of last year, she is a CIRM COMPASS undergraduate, a program which aims to increase equity within the field of regenerative medicine. Her interest in stem cells led her to focus on the use of human umbilical cords in current research, curious by its novel applications in this evolving field. She hopes readers will see the progress of stem cell research and how its future applications can be essential in treating various diseases that so many people are afflicted with each year.

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