Bruno Moises de Matos, Marco Augusto Stimamiglio, Alejandro Correa, Anny Waloski Robert
Abstract
Key Words: Pluripotent stem cells; Extracellular vesicles; Exosome; Cell-free therapy
Extracellular vesicles (EVs) are nanometric particles that are enclosed by a lipid bilayer and released by all cell types.They lack a functional nucleus and are therefore unable to replicate[1].EVs are composed of bioactive factors such as lipids, proteins, and nucleic acids, including mRNAs and non-coding RNAs[2].EV is an umbrella term that encompasses a heterogeneous population of membrane vesicles generated through a variety of mechanisms.The two major EV subpopulations include microvesicles(MVs) and exosomes (EXOs).EXOs are intraluminal vesicles of endosomal origin released when multivesicular bodies fuse with the plasma membrane, whereas MVs or ectosomes are generated from the outer budding of the plasma membrane[3].Due to their distinct biogenesis, MVs are generally larger(up to 1000 nm in diameter) than EXOs (less than 200 nm).However, these vesicle populations overlap not only in terms of size but also in composition[4].Recently, other nomenclatures were described in the“Minimal information for studies of extracellular vesicles 2018”guidelines (MISEV2018) based on the physical characteristics of EVs, for example, size (＜ 200 nm, small EVs; ＞ 200 nm, medium or large EVs)or density (low, middle, or high)[1].
Potential uses of EVs, such as for the diagnosis and treatment of pathologies or as potential drug carriers, have been investigated.In the field of regenerative medicine, the secretomes of adult stem cells,primarily mesenchymal stem/stromal cells (MSCs), including their EVs, are of great interest as they have been shown to act mainly in a paracrine manner rather than their potential for differentiation[5].An interesting list of advantages and disadvantages of the use of EVs instead of stem cells has been presented by Öztürket al[6].Among the advantages of using EVs cited by them and others are low immunogenicity and toxicity; minimal risk of malign transformation; minimal risk of getting trapped in the lung or causing vasculature obstruction; avoidance of contamination with undesired cell types;avoidance of uncontrolled cell division; the ability to manipulate EVs in order to obtain potential improvements; optimization of MSC culture to obtain a higher amount of EVs; and their ability to cross the blood-brain barrier, among others[4,6].In addition, EVs mimic the beneficial effects of MSCs in cell therapies in a wide range of animal models for different diseases[7-9].
MSC-derived EV (MSC-EV) has been extensively studied and has demonstrated several promising effects, as reviewed by Gowenet al[10], Tieuet al[11], Fuloriaet al[12], Kouet al[13], and Yudintcevaet al[14].However, despite the high potential of MSC-EVs, several factors limit their use.Recently some reviews highlighted the difficulty of establishing criteria to define the specific characteristics of MSC-EV and discussed the great variation in the MSC-EV preparations[15,16].Disadvantages of MSCs as a source for EVs include the variability between cells derived from different tissues, the variability between different donors, their limited ability to proliferate, the fact that they enter senescence, and genomic instability after a few passages[17].This raises the question of whether pluripotent stem cell(PSC) derived EVs have a similar to or better therapeutic potential than adult stem cell-derived EVs.
In this context, our objective is to show, using a non-systematic search, studies that use or characterize EVs derived from human PSC (hPSC-EVs) to understand the advances in the area.We also aim to identify the conditions in which the cells are cultivated for the isolation of EVs, how these are characterized, and any demonstrated applications (in vivoorin vitro).
Overview of hPSCs
hPSCs are characterized by unlimited proliferation and the potential to generate specialized cell lineages[18].Human embryonic stem cells (hESC) were first isolated from human blastocysts in 1998 by Thomsonet al[19], and to date, hundreds of hESC lineages have been established worldwide.hESCbased therapeutic technologies have applications in many diseases and conditions, such as spinal cord injuries, age-related tissue degeneration, and diabetes[20].However, ethical issues related to using cells from embryos have hindered the application of hESCs in research and treatment, leading to the development of the induced PSC (iPSC) technology by Takahashi and Yamanaka[21] and Takahashiet al[22].Since the generation of the first iPSC, many research groups have developed human iPSC (hiPSC)lineages reprogrammed from different adult cells, and obtained lineages very similar to hESC in terms of morphology and differentiation potential[23].For more information about hPSCs, see Karagianniset al[24], Liuet al[25], and Yamanaka[26].
Especially after the discovery of hiPSCs, pluripotent cells represented a promising alternative for regenerative medicine, transplants, disease modeling, and many other research applications[27-29].The possibility of generating pluripotent cells from patients and, from them, differentiated cells for tissue repair may mitigate common transplant issues, such as immunologic rejection.Nevertheless, the immunogenicity of pluripotent cells remains controversial[30], and the potential for tumorigenesis hinders the wide application of these cells in clinics.The risks of contaminating the differentiated cell populations with remaining pluripotent or proliferative cells, as well as the transmission of active pluripotency transcription factors or the acquisition of mutations by the pluripotent cells duringin vitroculture[26,31], limit the acceptance of hPSC-based therapies.Therefore, cell-free therapeutic approaches,including EVs, offer promising possibilities for applying hPSC-derived products[32].
It seems that the role of the secretomes of these cells has only recently begun to be investigated,possibly due to the difficulties still encountered in using hPSCs in the clinic.Some interesting studies show that EVs from ESCs could help with embryo implantation[33] and maintaining ESC stemness[34],while others have investigated the biogenesis of ESC-EVs[35,36], although they used murine PSCs.We will focus this review on studies with hPSCs due to their potential clinical applications.
HPSC-EVs: Isolation and characterization methodologies
The first investigation on the isolation of EVs from hPSC dates from 2015.In this initial approach, EVs were isolated from hiPSC cultured in Essential 8™ medium using differential centrifugation (DF)/ultracentrifugation (UC).It was shown that the hiPSC-derived EVs (hiPSC-EV) contain a variety of microRNAs (miRNAs) (such as miR-382, miR-611, and others) related to pathways such as focal adhesion, Wnt, PI3K-Akt, and MAPK signaling, as well as proteins related to processes involved in signal transduction, receptor binding, and others.In addition, the EVs positively affected the metabolism, proliferation, apoptosis rate, and differentiation capacity of cardiac MSCs.Βetter results were obtained when cells were exposed for only 22 h to EVs[37].This initial attempt demonstrated how hPSC-EVs could be beneficial and of interest for future acellular therapy applications.
Despite the potential of hPSC-EVs, we observed that the number of publications in this area is still low, and most of the existing publications date from the last five years (Figure 1A, Table 1).Some studies evaluate EVs that were isolated during the differentiation process or from cells that differentiated from PSCs, such as hiPSC-derived keratinocytes[38]; hPSC-derived cardiac progenitors or cardiomyocytes[39-41]; hPSC-derived MSCs[42-44]; hiPSC-derived neurons[45-47]; and hESC-derived chondroprogenitor cells[48].However, our review explores studies that isolated EVs from undifferentiated hPSCs.
Figure 1 Overview of studies on human pluripotent stem cell-derived extracellular vesicles published between 2012 and 2022.A: Timeline of published articles on human pluripotent stem cell-derived extracellular vesicles (hPSC-EVs).1Two articles were published online in 2022 but published in print in 2023; Β: Analysis of the percentage of articles that use human embryonic stem cells, human induced pluripotent stem cell, or both cell types to isolate EVs; C:Methods used in the studies to characterize hPSC-EVs.The graphic depicts the number of articles using certain techniques/total number of articles included in the analysis; D: Analysis of media used to culture hPSC to isolate the EVs.AFM: Atomic force microscopy; DLS: Dynamic light scattering; FC: Flow cytometry; NTA:Nanoparticle tracking analysis; qPCR: Quantitative polymerase chain reaction; SRM: Super-resolution microscopy; TEM: Transmission electron microscopy; TRPS:Tunable resistive pulse sensing; WΒ: Western blot; hESC: Human embryonic stem cells; hIPSC: Human induced pluripotent stem cell.
Table 1 Human pluripotent stem cell-derived extracellular vesicles: Methods of isolation and vesicle size
Using a non-systematic search, we found 36 studies that isolate hPSC-EVs mainly from the hiPSC lineages (Figure 1Β).Table 1 summarizes these studies, highlighting the cell culture medium used to culture the PSC, time of conditioned medium collection, EV isolation method, and EV mean size.The most common culture media were commercial, with defined components (Figure 1D).The two most common media used were mTeSR™1 (StemCell Technologies) and Essential 8™ medium (Thermo Fisher) (Table 1).A study published by Luoet al[49] aimed to optimize culture conditions for isolation of hiPSC-EVs.Using DMEM with different concentrations of EV-depleted KnockOut™ Serum Replacement (ED-KSR), they observed that cells remained viable at a 0.5% ED-KSR concentration and were able to isolate EVs from PSCs cultured in this condition efficiently.However, after 5 d of culture, there was a reduction in the expression of some pluripotency markers.Thus, although it may be cheaper than commercial media, it is necessary to consider the additional step of centrifugation of the KSR to remove particles, as well as the effects of the change in pluripotency-related parameters on the composition and potential of the EVs.
The biggest variations in EV isolation methods relate to the collection time of the conditioned medium: Many studies do not state the conditioning time.In most studies, however, the EVs were isolated after 24 h of cell culture or every 24 h for 3-5 consecutive days (Table 1), avoiding exceeding the 80%-90% cell confluence in the cell cultures.This collection time is possibly related to the nature of PSCs, as the culture medium must be changed daily, and cells must not reach 100% confluence to guarantee their viability and pluripotency.
Other relevant aspects of EVs are their size, morphology, and estimated particle concentrations.Most studies presented the information listed in MISEV2018, including positive and negative protein markers in EVs, usually using the western blot technique (31/36 articles) and performing a single EV analysis mainly using transmission electron microscopy (31/36 articles) to verify EV morphology and nanoparticle tracking analysis (20/36 articles) to verify their mean size and concentration (Figure 1C).The greatest number of studies used small EVs/EXOs, with sizes up to 200 nm (small EVs) (Table 1).
The most common method for hPSC-EV isolation is DF (here defined as the initial centrifugations to remove cellular debris and apoptotic bodies) followed by UC (Table 1).Although this is the most common method used, it is unsuitable for isolating EVs from large-scale experiments and clinical trials.Using a large-scale 2D culture, Andradeet al[69] isolated hPSC-EVs using tangential flow filtration(TFF) with or without subsequent UC (TFF + UC).The isolated EVs presented a size of approximately 100 nm, regardless of whether UC had been performed, with similar particle concentration, although TFF + UC resulted in a smaller number of proteins.The effect of different culture conditions (hypoxia -1% O2, physiological hypoxia - 5% O2, and normoxia) on the therapeutic potential of hPSC-EVs was also
investigated.The results showed that EVs derived from hPSC cultured in 1% O2(hypoxia) had greater angiogenic potential than those derived under other conditions and that better results were achieved when obtaining EVs using TFF[69].
Another highly discussed topic about PSCs is the possible formation of teratomas, as well as the biodistribution of these cells when applied inin vivomodels.These concerns also extend to PSC-EVs.To clarify these points, Guet al[75] evaluated the safety and biodistribution of hiPSC-EVs.They used several approaches to show that PSC-EVs are safe, have no adverse effects on cells (e.g., do not cause hemolysis), are not genotoxic, and can be administered by different routes (nasal, intramuscular, or intravenous) without generating adverse effects (e.g., inflammation at the site or pathological changes in the organs of rats).
Potential therapeutic applications of hPSC-EVs
Although few investigations have been carried out with hPSC-EVs, we notice that almost all of them have already applied hPSC-EVs to different disease models, bothin vitroandin vivo.PSC-EVs have been described as having: Protective effects inin vitroandin vivomodels of ischemia-reperfusion kidney injury[64]; neural protective abilities[60]; the capacity to modulate neuroinflammation and protect against ischemic stroke through Treg cell expansion[73]; antifibrotic effectsin vivoand inin vivomodels of liver injury[61,72]; and reduced cartilage degradation in an osteoarthritis model[77].They have shown improvements in wound closure, angiogenesis, and increased nerve fiber density in a woundhealing diabetic mouse model[54,79]; and improved recovery of ovarian function in a premature ovarian failure mouse model[67].EVs were also associated with acellular nerve grafts, demonstrating their potential to repair peripheral nerve defects[80].
It was also demonstrated that MVs, but not EXOs, dedifferentiated Müller cells into retinal progenitor cellsin vitro[71].Other studies showed the ability of PSC-EVs to promote regeneration of diseased or damaged retinas[56] and to accelerate corneal epithelium defect healingin vivo[68].Other potential uses cited for PSC-EVs are: In antitumoral activity[51,63]; in angiogenesis stimulation[69]; as a gene delivery vector[50]; to increase the functional properties of cord blood-derived hematopoietic stem and progenitor cells[70]; and to improve the number of beating EΒs depending on the hiPSC origin[66].
One noteworthy effect shown in some studies is the capacity of PSC-EVs to “rejuvenate”different cell types, such as senescent endothelial cells[52,58], senescent human dermal fibroblasts[55], senescent chondrocytes[77], and others.Considering this potential, the hPSC-EVs, hESC-EVs, and hiPSC-EVs were investigated as therapeutic tools for age-related diseases.Regarding neurological diseases, the hPSCEVs showed potential in recovery of senescent hippocampal neural stem cells in rats with vascular dementia - partially through the transfer of miRNAs that inhibit mTORC1 activation - resulting in an improvement in disease status (e.g., reverse cognitive impairment)[81].Furthermore, using mice of varying ages, hPSC-EVs were found to rejuvenate hippocampal neural stem cells partly through the transfer of SMAD proteins that activate myelin transcription factor 1 (MYT1), which is reduced in senescent cells, and activates a signaling cascade in the MYT1-Egln3-Sirt1 axis[81].
In an ischemic stroke model, hPSC-EVs reduced the expression of inflammatory cytokines and leukocyte infiltration, and increased the number of regulatory T cells and other immunomodulatory effects that alleviate neurological deficits[73].They also reduced blood-brain barrier damage in aged stroke mice through blood-brain barrier rejuvenation, partially through the transfer of AKT1 and CALM from EVs to endothelial cells leading to activation of the endothelial nitric oxide synthase-Sirt1 axis[78].Therefore, hPSC-EVs could be a promising cell-free therapy to treat age-related diseases associated with cellular senescence.
In order to evaluate the benefit of hPSC-EVs compared to other EVs, one interesting study demonstrated that both hiPSC-EVs and hMSC-EVs, isolated through size exclusion chromatography(Table 1), could improve the proliferation of senescent MSCs and alleviate cellular aging in a replicative aging model, possibly modulating reactive oxygen species production with peroxiredoxins presented in EVs.However, despite the similar effects, EVs derived from iPSCs enter target cells more efficiently,and the production of hiPSC-EVs was about 16-fold higher than that of MSC-EVs (using the same culture medium)[59].
hPSC-EV composition
Even though many articles described the effects of hPSC-EVs, few made deeper characterizations of, for example, the protein and miRNA content of these EVs.Some performed proteomic analysis to help explain some of the effects[59] or as a control (time 0) to study the differentiation process[39,46].In one interesting approach using high-density lectin microarray, Saitoet al[57] demonstrated that rΒC2LCN, a specific lectin for hPSCs, bound to hiPSC-derived EVs but not to adipose-derived stem cell-, hemodiafiltration-, or chondrocyte-derived EVs, which suggests a particular glycan-signature for hiPSC-EVs,resembling the glycome signature of the cell surface.
One recent study that provided a detailed description of the contents of hPSC-EVs was conducted by Βiet al[74].The proteomics of hESC-, hiPSC-, and hMSC-EXOs showed that the main enriched proteins were related to distinct pathways between vesicles of pluripotent and multipotent cells.In hPSCs, EXO content was more focused on development, metabolism, and anti-aging properties, and in hMSCs, it was related to immune regulation.Another study in 2022 also indicated that hMSC-EV content is strongly related to immune regulation while hPSC-EV content does not present many of the proteins related to this function[76].Actually, 79 proteins were found to be shared between hMSC- and hPSCEVs, yet the main biological processes related to them were DNA regulation, signal transduction and cell communication[76].Liuet al[59] also compared the protein content of hiPSC-EVs and hMSC-EVs and described more than 1100 proteins shared between the different EVs, allowing to identify proteins that could be responsible for the anti-senescent effect observed in the study.
Considering the protein content of hESCs and hiPSC-EXOs, Βiet al[74] suggested that hESC-EXOs are more prone to regulate development and pluripotency pathways, and hiPSC-EXOs have a stronger correlation with metabolism.Regarding the most enriched miRNAs for both hPSC-EVs, it was shown that they were related to cell cycle and metabolism regulation.Interestingly, miRNAs found in both hESC-EXOs and hiPSC-EXOs were involved in cell differentiation, development, and cell cycle, even though the hiPSC-EXO set of miRNAs seemed to play a less significant role in these functions than the hESC-EXO set[74].
In order to explore whether apoptosis-linked gene 2-interacting protein X (ALIX), a protein present in the endosomal sorting complex required for the transport and biogenesis of EXOs, could regulate the protein content of EV, Sunet al[62] isolated EVs from hiPSCs in which ALIX was overexpressed (using lentiviral transduction) or were knocked out (using CRISP-Cas9 system).EVs isolated from these cell lineages were of similar size, although EVs generated from knockout cells were slightly larger.The evaluation of protein content in EVs showed that those derived from knockout cells had fewer proteins,while EVs from overexpressing cells presented a higher number of proteins.These differences could be related to the differences demonstrated in functional assays,e.g., cell viability, apoptosis inhibition, and formation of capillary-like structures, where EVs from overexpressing cells had better effects.So, EVs with different protein profiles could have different therapeutic applications.
Although hPSC cultivation has been carried out for some time, the requirements forin vitroculture of these cells are very specific, as many factors are necessary to maintain them in their undifferentiated state.This, together with the cost, could be one of the reasons why secretomes and isolation of hPSCEVs have not been extensively studied so far.Commercial media are now defined with a few components that are no longer as expensive as before, which may have contributed to the increase in publications in recent years.
An overview of the hPSC-EV studies is shown in Figure 2, which illustrates the potential use of these EVs for regenerative medicine.Regarding EV characterization, we observed in the publications that hPSC-EVs follow the basic requirements described in MISEV2018.However, despite the recent increase in research in this area, further characterization of the content of these EVs needs to be carried out.In addition, studies with modified cells aimed to enrich the content of EVs with some specific protein or miRNA may be of great interest.One interesting approach requiring more extensive discussion is the possible use of hPSC-EVs in reprogramming adult cells into PSCs.A recent study used EVs derived from ESCs undergoing cardiac differentiation to transdifferentiate fibroblasts to cardiomyocyte-like cells with relatively high efficiency[82].
Figure 2 Diagram of pluripotent stem cell-derived extracellular vesicle isolation, its most common characterizations, and the applications described or indicated for these extracellular vesicles.EV: Extracellular vesicle; miRNA: MicroRNA; lncRNA: Long noncoding RNA; PSC: Pluripotent stem cell.The images were obtained from Servier Medical Art (http://smart.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License.
Our review shows that hPSC-EVs have therapeutic potential, although no publications demonstrate that they are effectively better than other EVs, such as hMSC-EVs.hPSC can be obtained from different sources (embryonic or reprogrammed from adult cells) and, despite showing some heterogeneity between lineages, they are highly similar in their main characteristics: They are pluripotent and with a high proliferative capacity.The latter makes it possible to obtain a large number of EVs.It should be noted that PSC-EV derived from different hPSC lineages may show some variability in their content.Βut considering the fact that we can isolate EVs from a single source (a homogenous culture), this can possibly bring less variability between batches compared to other common EV sources.However,studies in this area are still needed as current results are highly variable.Alternatives to EVs include the use of cell-engineered nanovesicles generated by serial extrusion of hiPSCs, as described by Leeet al[83], which presented similar results to PSC-EVs, but with higher production yield.However, more studies are needed to verify the viability of this method for future applications.Thus, challenges that remain are the large-scale production of EVs, which in the case of hPSC cultivation can be expensive,and the investment in efficient methodologies for EV isolation that could be used in good manufacturing practices for future acellular therapies.
Author contributions:All authors contributed in literature review and wrote the manuscript; Stimamiglio MA revised the manuscript; Correa A and Robert AW designed the research; and all authors have read and approved the final manuscript.
Conflict-of-interest statement:All the authors report no relevant conflicts of interest for this article.
Open-Access:This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC ΒYNC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is noncommercial.See: https://creativecommons.org/Licenses/by-nc/4.0/
Country/Territory of origin:Βrazil
ORCID number:Bruno Moises de Matos 0000-0002-9868-7044; Marco Augusto Stimamiglio 0000-0001-8537-2812; Alejandro Correa 0000-0002-5887-1453; Anny Waloski Robert 0000-0002-7837-2790.
S-Editor:Wang JJ
L-Editor:Wang TQ
P-Editor:Guo X
