Clinical Relevance of Mesenchymal Stromal Cells from Various Sources: Insights into Transcriptome Analysis for Identifying Inherent Potential

Abstract

This book chapter provides an in-depth overview of the clinical relevance of mesenchymal stem cells (MSCs) derived from various sources, highlighting the importance of whole transcriptome analysis in revealing their inherent potential. The chapter delves into different sources of MSCs, such as bone marrow, adipose tissue, umbilical cord, and placenta, and compares their respective properties and capabilities. Additionally, it explores the latest advancements in whole transcriptome analysis, including RNA sequencing and microarray analysis, and their applications in MSC research. The aim is to provide a comprehensive understanding of how high-end technologies, such as whole transcriptome analysis, can aid in identifying the inherent potential of cells for therapeutic applications. It will also discuss how such gene expression approaches helped identify the inherent potential of specific MSC sources, tailoring their use towards specific clinical applications, including immune tolerance and modulation, osteogenesis, and chondrogenesis. Additionally, it highlights the importance of extracellular vesicles derived from MSCs. This knowledge will be beneficial for researchers and clinicians working towards developing MSC-based treatments for regenerative medicine and cellular therapy.

1. Introduction

Mesenchymal stromal cells (MSCs), also referred to as mesenchymal stem cells, are multipotent stromal cells with self-renewal capacity and multilineage differentiation potential [1]. There has been an increasing interest in MSCs in recent years due to their unique properties, including long-term proliferation, multilineage differentiation potential, and immunomodulatory capabilities [1]. These properties make MSCs a promising candidate for use in regenerative medicine and cellular therapy.

The discovery of MSCs dates back to 1976, when Friedenstein et al. identified a group of non-hematopoietic, plastic-adherent, fibroblast-like cells that could be extracted from the bone marrow (BM). Since then, MSCs have been identified in various tissues, including adipose tissue (AT), umbilical cord (UC), and amniotic fluid [2, 3]. It is, however, essential to note that MSCs are not homogenous in their properties and capabilities. The source from which they are derived significantly determines specific cellular characteristics. MSCs from different tissues exhibit distinctive properties that may influence their potential clinical applications [4, 5]. Several studies have been conducted that compare MSCs from various sources [6, 7, 8]. For instance, Hsieh et al. performed a side-by-side functional comparison between UC-MSCs and BM-MSCs and found that BM-MSCs can be easily differentiated into osteocytes compared to UC-MSCs, which showed delayed and insufficient differentiation into osteocytes [8]. Another study by Karahuseyinoglu et al. identified that UC-MSCs possess higher chondrogenic differentiation capacity compared to BM-MSCs [9]. Therefore, it is critical to understand the inherent potential of MSCs from various sources before they can be used for clinical applications.

Whole transcriptome analysis is a powerful tool that enables researchers to gain a deep understanding of the gene expression patterns and regulatory networks that govern the properties and functions of MSCs. This cutting-edge approach allows for a comprehensive exploration of the molecular mechanisms that underlie the unique characteristics of MSCs. To this end, various studies have analyzed the global gene expression profile for different types of MSCs [10, 11, 12]. Some of these studies focused on evaluating the expression levels of pluripotency genes and embryonic stem cell (ESC) markers [13, 14]. Other studies investigated the expression of genes that have functional significance, such as genes involved in bone and cardiovascular development [13, 15]. Overall, whole transcriptome analysis is a valuable tool for gaining insights into the complex mechanisms that govern the behavior of MSCs.

This chapter will review the clinical relevance of MSCs derived from various sources, highlighting the importance of whole transcriptome analysis in unraveling their inherent potential. It will discuss the different sources of MSCs, including BM, AT, UC, and placenta (PL), and compare their properties and capabilities. Additionally, it will explore the latest developments in whole transcriptome analysis, including RNA sequencing and microarray analysis, and their applications in MSC research. The aim is to provide a comprehensive insight into high-end technologies, such as whole transcriptome analysis, and how they can aid in identifying the inherent potential of cells for therapeutic applications. This knowledge will be useful for researchers and clinicians working towards developing MSC-based treatments for regenerative medicine and cellular therapy. Furthermore, new strategies to enhance the therapeutic potential of MSCs are discussed.

2. Mesenchymal stem cell sources

While bone marrow-derived MSCs (BM-MSCs) have been extensively studied for the past four decades, the last decade has witnessed a shift towards exploring alternative sources of MSCs. This shift is driven by the search for cell sources that are easily accessible, noninvasive, and available in abundant quantity [16]. As a result, MSCs have been successfully isolated from diverse tissues, such as AT and perinatal tissues like the UC and PL [2, 3, 17]. Each source of MSCs has its advantages and limitations, and ongoing research aims to further understand their unique properties and refine their applications in clinical settings. Additionally, studies are exploring other sources, such as synovial fluid, dental pulp, and more, expanding the potential repertoire of MSCs for therapeutic purposes. Besides the MSCs derived from the BM, other tissue sources of MSCs provide technical advantages. For instance, harvesting BM-MSCs involves an invasive procedure, and their expansion is imperative due to the restricted number of stem cells [18]. Conversely, adipose tissue-derived MSCs (AT-MSCs) are more accessible and produce a greater number of cells, simplifying the expansion process [19]. Such technical distinctions highlight the importance of tailoring protocols for isolating and expanding MSCs based on their specific tissue source.

2.1 Bone marrow-derived MSCs (BM-MSCs)

Traditionally considered the gold standard, BM-MSCs have been the focus of extensive research and clinical trials [20]. Isolation from the bone marrow involves a relatively invasive procedure, typically bone marrow aspiration from the iliac crest [21]. BM-MSCs exhibit robust self-renewal capacity and the ability to differentiate into various cell lineages, including osteoblasts, adipocytes, and chondrocytes [22]. Surface markers commonly associated with BM-MSCs include CD90, CD105, CD73, and CD44, while they lack the expression of hematopoietic markers such as CD34 and CD45 [23]. Research on the application of BM-MSCs has shown promising results in treating various diseases [24]. Nonetheless, discrepancies in results among studies can be ascribed to factors like donor variability, cell preparation procedures, and a deficiency in standardization before transplantation [25]. It is important to note that the expansion and cultivation process can vary based on specific protocols, and researchers continuously work on optimizing these procedures for the best outcomes in terms of cell yield, quality, and therapeutic efficacy.

2.2 Adipose tissue-derived MSCs (AT-MSCs)

AT has emerged as a rich and easily accessible source of MSCs. AT-MSCs can be isolated from fat tissue through a minimally invasive procedure, usually involving liposuction or fat removal during other surgical procedures. AT-MSCs share similarities with BM-MSCs in terms of their differentiation potential [26, 27]. However, they may offer advantages in terms of abundance and ease of isolation, making them an attractive alternative to regenerative therapies. As BM-MSCs, AT-MSCs express typical stem cell surface markers and possess the potential to differentiate into various lineages [23, 28]. Due to its accessibility and abundance, AT yields the isolation of stem cells at a quantity 500 times greater than those obtained from bone marrow [29]. AT-MSCs have been studied for their ability to enhance wound healing processes. The cells contribute to tissue regeneration by promoting the proliferation and migration of various cells involved in wound repair [30, 31, 32]. Other studies have investigated the potential of AT-MSCs in neural tissue repair. AT-MSCs may contribute to neuroprotection and neurodegeneration after injury [33]. They have also demonstrated a pro-angiogenic effect, promoting the formation of new blood vessels. Studies suggest that the paracrine factors released by AT-MSCs contribute to angiogenesis, which is crucial for tissue repair and regeneration [34]. Research and preclinical studies have explored the use of AT-MSCs in specific autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus. Clinical trials have been conducted or are ongoing to assess the safety and efficacy of AT-MSCs in treating these conditions [35]. However, additional research is crucial to establish the optimal dosage, administration route, and safety parameters for clinical applications [36].

2.3 Umbilical cord-derived MSCs (UC-MSCs)

MSCs derived from the UC present a unique perinatal source with distinct properties. Isolation from the Wharton’s jelly of the UC is considered noninvasive and ethically uncontentious. UC-MSCs exhibit multilineage differentiation potential, like BM-MSCs. Moreover, they possess immunomodulatory properties that make them particularly interesting for therapeutic applications, especially in the context of immune-related disorders [24, 37]. In 1991, McElreavey et al. [38] made a groundbreaking discovery by isolating fibroblast-like cells from the Wharton’s Jelly (WJ) of the human UC. These cells are readily available after childbirth, providing a noninvasive and ethical source of MSCs. Obtaining a significant quantity of UC-MSCs through several passages and extensive ex vivo expansion is a simple process [39]. UC-MSCs are considered to be in a more primitive state compared to MSCs from other tissues, and they possess a high proliferative capacity [40]. In addition, UC-MSCs can be cryopreserved and stored for future use, allowing the creation of cell banks for potential therapeutic applications [41]. UC-MSCs display comparable surface phenotypes, adherence to plastic surfaces, and multipotency characteristics as MSCs obtained from other MSC tissue sources. UC-MSCs present versatile applications in both autologous and allogeneic contexts. Autologous use involves employing UC-MSCs for the same individuals they are sourced from. On the other hand, allogeneic use refers to the application of UC-MSCs across different individuals. Allogeneic UC-MSCs can be expanded and cryopreserved in cell banks, ready for use by patients in need. However, it is crucial to verify the health of the baby, who is the donor of UC-MSCs, through genomic or chromosomal tests, as their normal growth without health problems cannot be guaranteed in advance. Understanding the advantages and disadvantages is essential for each specific clinical application in both autologous and allogeneic scenarios [42].

2.4 Placenta-derived MSCs (PL-MSCs)

The PL plays a crucial role in fetal development by providing nutrition and supporting immunological tolerance. In recent times, stem cells derived from the PL, known as PL-MSCs, have gained attention as an alternative perinatal source of MSCs in regenerative medicine. PL-MSCs, originating from the fetus, exhibit the capacity for self-renewal and multipotency, possessing immunomodulatory properties, making them valuable for therapeutic applications [43]. Additionally, the PL harbors a substantial number of MSCs, and its utilization for research is not encumbered by the ethical concerns associated with human embryonic stem cells [44, 45].

2.5 Other sources of MSCs

2.5.1 Dental pulp-derived MSCs (DP-MSCs)

The dental pulp is the soft tissue at the core of a tooth, a highly vascularized connective tissue. It is enveloped by mineralized hard tissue and contains diverse cell types, including odontoblasts and undifferentiated progenitor cells [46]. Within this population of undifferentiated progenitors, MSCs are found to exhibit a high proliferation rate and a high degree of multipotency [47, 48, 49]. Adherent colonies of spindle-shaped cells, known as Colony-Forming Units Fibroblast (CFU-F), are formed by human dental pulp-derived stem cells. The analysis of CFU-F reveals that human dental pulp harbors a more abundant population of MSCs compared to human bone marrow. Specifically, the CFU-F capacity of DPSCs is five times greater than that of BM-MSCs [50, 51]. DP-MSCs have shown great promise in dental applications. They held the potential for dental pulp regeneration [52] and dentin repair [53], and showed regenerative capacity for nerve repair [54].

2.5.2 Synovial fluid-derived MSCs (SF-MSCs)

The existence and attributes of MSCs in synovium specimens were initially documented by De Bari et al. [55], and subsequent studies have extensively explored this topic [56, 57]. MSCs derived from synovium specimens (SF-MSCs) exhibit superior proliferative capacity and chondrogenic potential compared to MSCs from other sources. Various studies have demonstrated the multilineage differentiation potential of SF-MSCs in humans [58, 59]. Recent findings indicate that the chondrogenic differentiation capacity of equine SF-derived MSCs is comparable to that of bone marrow MSCs [60, 61], confirming the stemness of these cells according to the criteria set by the International Society for Cellular Therapy [23].

3. Transcriptome analysis and identification of inherent potentials

MSCs are being actively used in clinical trials as potential cellular therapies for various clinical conditions, with over 1000 trials registered on ClinicalTrials.gov (http://www.clinicaltrials.gov). MSCs isolated from the bone marrow are the first to be used in clinical trials and are the most commonly studied MSC source in preclinical and clinical studies [62]. MSCs from other sources are also being used to treat different clinical conditions, including MSCs from AT, UC, and others. However, variations in the clinical outcomes of MSC treatments exist, and to date, there is no consensus on the most suitable MSC source to treat a specific disease [63]. The inconsistent clinical outcomes of MSC treatments can be attributed to the heterogeneous potency and functional variations of the MSCs and the lack of efficient assays for assessing their potency [7, 62].

MSCs are still identified by their expression of a specific subset of surface marker proteins, trilineage differentiation potential, and immunomodulatory potential [23, 64]. Although these criteria have served the research community for a long time, they do not represent the heterogeneity of the MSC population. The scientific community should benefit from advanced technologies, such as whole transcriptome analysis and single-cell RNA-seq technology, to explore MSC identity and function regarding their tissue origin and functional status.

These approaches could provide a comprehensive view of gene expression patterns in MSCs, enabling researchers to gain a deeper understanding of the inherent capabilities of MSCs, including their differentiation potential, immunomodulatory effects, and regenerative properties. In a prior study, we conducted a gene expression analysis of four types of MSCs, including MSCs derived from the BM, AT, UC, and PL [16]. Our analysis revealed a unique set of genes that are either up- or down-regulated in one type of MSC compared to all other stem cell types. Moreover, we identified signature genes exclusively expressed in one type of MSC [16]. These findings clearly indicate intrinsic differences in the potential of MSCs from different sources.

Such information can be leveraged to develop more effective and targeted therapies for various conditions, such as osteoarthritis, cardiovascular disease, and neurological disorders. Moreover, whole transcriptome analysis can identify the specific genes and pathways involved in the biological processes of MSCs. Such an approach can refine the operational definition of MSCs and further our understanding of their native physiological function. Furthermore, this knowledge can inform the development of optimized culture conditions and clinical manufacturing protocols, leading to more consistent and effective MSC-based patient therapies. In the following sections, we will discuss how such gene expression approaches helped identify the inherent potential of specific MSC sources, tailoring their use towards specific clinical applications, including immune tolerance and modulation, osteogenesis, and chondrogenesis.

3.1 Immune tolerance and modulation

The modulation of the immune system and its tolerance is a complex process involving gene expression related to various biological functions. One of the critical properties of MSCs is their immune tolerance and survival mechanisms, which are essential for the success of stem cell transplantations in treating diseases [65]. MSCs typically express major histocompatibility (MHC) Class I antigens on their surface and not Class II; however, Class II antigens are upregulated by inflammatory agents [66]. Studies have shown that both autologous and allogenic MSCs prevent lymphocyte proliferation without causing apoptosis of T cells [67, 68]. MSCs release factors such as transforming growth factor β (TGF-β), interleukin 10 (IL-10), interleukin 6 (IL-6), and nitric oxide, which are known to affect immune cells [69, 70]. MSCs also affect the maturation of immune cells, increasing regulatory T cells (Treg), anti-inflammatory T helper 2 (TH2), and dendritic (DC2) cells [66, 71, 72]. Furthermore, they were found to induce M1 macrophages to adopt the anti-inflammatory M2 form and reduce IgG production from B cells [73]. Although the immunoregulatory potential of MSCs has been well established, the mechanisms underlying their actions are not yet fully understood [65]. Moreover, variations in the immunomodulatory capabilities among different types of MSCs are identified [6, 74]. These findings are manifested not only in in vitro studies but also in the outcomes of various clinical trials. For example, AT-MSCs were found to possess greater immunosuppressive capabilities than BM-MSCs in vitro and in vivo [75, 76].

Whole-gene expression analysis of MSCs from various sources can help identify the most suitable cells for clinical use by uncovering differences in their immunomodulatory potential. We previously conducted a comprehensive transcriptome analysis of MSCs from four sources to gauge their inherent potential [16]. We studied around 1400 immune modulation-related genes to understand if there were any differences in the MSCs’ ability to modulate immune responses. Our findings indicate that toll-like receptor 4 (TLR-4) expression was highest in BM-MSCs and lowest in UC- and PL-MSCs. While TLR-4 is traditionally known for activating innate immune cells against pathogens, its activation in MSCs has also been discovered to facilitate interactions with the surrounding environment [77] and induce Treg cell activation, which counteracts the inflammatory aspect of several diseases [78]. We have identified a significant immune modulatory trend in BM-MSCs, which was emphasized with increased expression of interleukin 7 (IL-7), an inhibitor of T-cell proliferation [79]. We also found an increase in CD200 expression, an immune player, in both BM- and UC-MSCs compared to other MSC types. In addition to its immune tolerance roles, CD200 inhibits the maturation of myeloid progenitors into inflammatory cells and suppresses the secretion of proinflammatory TNF-α in stimulated macrophages [80, 81]. On the other hand, CD274, a known immune-modulatory protein that plays a negative role in immune modulation, was found to be uniquely upregulated in UC- and PL-MSCs. Additionally, we found several proinflammatory pathways to be activated in other MSC types compared to BM-MSCs, such as interleukin 8 (IL-8) signaling, diabetes mellitus signaling, and TWEAK signaling. Our transcriptome analysis suggests that BM-MSCs may have a higher immune modulation potential than other MSC types.

Several studies have been conducted to evaluate the similarity and variability of gene expression in MSC samples [11, 82, 83]. In a study by Sun C et al., the transcriptomic variation of MSCs from different tissues, including PL, UC, and dental pulp, was investigated in relation to immunomodulatory function [10]. They found that among the genes that exhibited highly variable expression were genes involved in the differentiation process of MSCs and genes that regulated immunomodulation, such as CD274, C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), IL6, colony-stimulating factor 3 (CSF3), and hepatocyte growth factor (HGF). Conversely, genes involved in other biological processes, such as metabolic processes, gene expression, RNA processes, and RNA binding, showed minimal gene expression changes. The study also examined the transcriptomic changes of INFγ-preconditioned MSCs to identify the molecular mechanisms behind the varying immunomodulatory potencies of different MSC samples. They found that different groups of MSCs use similar regulation networks in response to inflammatory stimulations, but gene expression variations within those networks result in differences in immunosuppressive capability. These gene expressions were found to vary greatly between MSC samples. Accordingly, the study identified a panel of these responsive genes that can be used to assess the immunosuppressive potency of MSCs.

Generally, whole transcriptome analysis studies enable investigation of the cause of immunomodulatory functional variation in different MSC types at the molecular level. Consequently, it can aid in establishing minimum clinical release criteria in our pursuit to identify the best MSC source with immunomodulatory potency.

3.2 Osteogenesis and chondrogenesis

In accordance with the guidelines set forth by the International Society for Cellular Therapy (ISCT), human MSCs are required to possess the ability to differentiate into three distinct lineages in vitro, namely osteoblasts, adipocytes, and chondroblasts [23]. While all MSCs from different tissue sources must be able to demonstrate osteogenic and chondrogenic differentiation capabilities, variations in their differentiation potential have been extensively studied and reported [8, 84, 85, 86]. These inherent differences have significant implications, particularly when deciding on the source of MSCs to be used in clinical trials for related medical conditions such as cartilage regeneration in patients with knee osteoarthritis. The selection of appropriate MSCs is crucial to ensuring successful outcomes in such clinical trials and the development of effective therapies.

Our previous study, which analyzed the global gene expression profile of MSCs from different sources, revealed that BM-MSCs and AT-MSCs exhibit a higher expression of genes related to osteogenesis. We identified forkhead box C1 (FOXC1) and distal-less homeobox 5 (DLX5) among the upregulated genes in BM-MSCs. FOXC1 plays a crucial role in regulating initial osteoblast differentiation by directly regulating the expression of Msx2, a key regulator of early osteogenic events [87]. Similarly, DLX5 is a transcription factor that plays a role in later stages of osteogenic differentiation and is regulated by bone morphogenic protein 2 (BMP2) signaling. On the other hand, AT-MSCs showed upregulation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene, which binds to BMP ligands and transduces BMP signaling [88]. These results suggest that both BM- and AT-MSCs have a higher capacity to differentiate into osteoblasts than PL-MSCs and UC-MSCs. Our findings are consistent with other studies that analyzed gene expression profiles of MSCs from various sources, which also demonstrated that BM-MSCs have better osteogenic potential than MSCs from other sources [8, 89]. It is worth noting that several studies have reported comparable osteogenic differentiation capabilities of MSCs derived from different sources, such as BM, UC, or AT [90, 91]. However, in this discussion, we are primarily concerned with the inherent osteogenic potential that undoubtedly reflects the osteogenic differentiation capacity of MSCs, although it does not exclude the differentiation capacity of MSCs obtained from other sources.

A recent study conducted by Zhang et al. utilized advanced single-cell RNA sequencing to investigate the similarity and heterogeneity of BM-MSCs and MSCs derived from Wharton’s Jelly [92]. The study revealed a unique gene expression profile for each MSC cell source and identified heterogeneity among MSCs from the same tissue source, with distinct subpopulations of cells being identified. These subpopulations were categorized according to their gene expression patterns. Notably, the study identified a multipotent progenitor subpopulation that had an expression signature enriched for trilineage differentiation, including osteogenic and chondrogenic differentiation. However, the study did not draw any conclusions about which cell source possesses a higher osteogenic or chondrogenic differentiation capability. This study provides valuable insights into the inherent cellular composition of MSCs from different sources. The unique gene expression profiles identified for each cell source highlight the potential differences in their therapeutic applications. Furthermore, the identification of a multipotent progenitor subpopulation with an expression signature enriched for trilineage differentiation is of particular interest for regenerative medicine applications.

The ability of MSCs to differentiate into chondrocytes has made cartilage repair a successful regenerative application of these cells. Among the several bone and cartilage disorders investigated in MSC-based clinical studies, knee osteoarthritis is one of the most extensively studied, with various clinical trials employing MSCs from different sources [93, 94, 95]. Most of these trials have utilized an autologous source of MSCs, either from the BM or AT, and have produced encouraging results, with one study progressing to the phase III stage [96]. Other sources, such as WJ, PL, and amniotic membrane/fluid, have also been explored [96]. Evaluating the inherent chondrogenic and survival potential of the MSC type before its use in clinical settings could minimize the risk of suboptimal cell sourcing, leading to improved patient outcomes.

We have conducted a whole transcriptome analysis of four tissue-specific MSCs and evaluated the expression level of 43 genes related to chondrogenesis [16]. Our results revealed that BM- and AT-MSCs exhibited the highest number of upregulated genes. In BM-MSCs, chondroitin Sulfate N Acetylgalactosaminyltransferase 1 (CSGalNAcT-1), chitinase-3-like 1 protein (CHI3L1), and mothers against decapentaplegic homolog 1 (SMAD1) were specifically upregulated. CSGalNAcT-1 plays a crucial role in initiating cartilage chondroitin sulfate biosynthesis [97], while CHI3L1 is associated with proliferation and differentiation in osteogenic and chondrogenic cell lineages during fetal development and MSC differentiation [98]. SMAD1 is an essential regulator of Smad-dependent signal transduction pathways, which is vital for initiating chondrogenic differentiation [99]. In contrast, transforming growth factor beta-induced (TGFBI), transforming growth factor beta receptor 2 (TGFBR2), and short stature homeobox 2 (SHOX2) were found to be downregulated in all MSCs compared to AT-MSCs. Research has shown that transforming growth factor beta (TGFB) proteins are the most potent activators of chondrogenesis in human MSCs [100]. Inactivation of TGFBR2 in neural crest cells resulted in the aberrant formation of Meckel’s cartilage and altered the development of the mandible [101]. SHOX2 has been shown to control chondrocyte maturation by regulating the expression of Runt-related transcription factor 1 (RUNX) genes through bone morphogenic protein 4 (BMP4) [102]. Therefore, our results propose that BM and AT-MSCs have higher chondrogenic differentiation potential than PL and UC-MSCs. Such findings provide a deeper understanding of the molecular mechanisms involved in chondrogenic differentiation and could potentially inform the development of new regenerative therapies for cartilage repair.

3.3 Extracellular vesicles of MSCs and exosome potential

Besides direct cell-to-cell contact, the therapeutic potential of MSCs is attributed mainly to their paracrine actions [103, 104]. These actions are primarily mediated by the secretion of extracellular vesicles (EVs), particularly exosomes. Exosomes are small EVs with an average size of ~100 nm that are released by the cells into the extracellular environment [105]. They are enriched with bioactive molecules, such as nucleic acids, proteins, and metabolites, with various important biological functions [106]. Exosomes play a crucial role in intercellular communication and can transfer important biomolecules to target cells, thereby modulating various physiological and pathological processes [107, 108]. Studies have demonstrated that EVs of MSCs can exert biological effects comparable to MSCs themselves [109, 110]. As such, the paracrine effects of MSCs through EV secretion have emerged as a promising avenue for developing novel cell-free therapies for multiple diseases.

The unique advantage of EV as an alternative therapy for MSCs stems from the fact that they are cell-free therapies and accordingly require less authority approval due to their superior safety profile and lower immunogenicity [111, 112]. In addition, the viability, longevity, and entrapment of MSCs in the lung microvasculature after implantation hinder their use [113, 114, 115], whereas EVs of MSCs do not follow these limits [103]. Although the therapeutic potential of MSCs-EV has been extensively studied in vivo, its applications in a clinical setting have been poorly investigated [111, 116]. Only a few clinical studies have demonstrated the effectiveness of their use [117, 118, 119].

Given the high potential of exosomes in modulating cellular behaviors and their crucial role in the paracrine action of MSCs, it is imperative to gain a deeper understanding of their function to develop effective exosome-based therapeutics. It has been observed that exosomes derived from different MSC types have varying effects, and different culture conditions also result in exosomes with greater therapeutic effects [103, 120]. Transcriptome analysis of exosome cargo can aid in elucidating key factors that govern their biological activities. Accordingly, such data could lead to the development of more effective therapies. For instance, Yao et al. investigated the effect of exosomes from BM-MSCs on pancreatic cancer cell characteristics [104]. They performed transcriptome sequencing on the BM-MSCs-derived exosomes at different stages and identified, for the first time, the role of circular RNA (circRNA) found in the BM-MSCs-derived exosomes in pancreatic cancer. The circRNA (circ_0030167) was found to be enriched and identified as the main effector molecule of the BM-MSCs-derived exosome. From there, they identified that circ-0030167 inhibited the malignant progression and stemness of pancreatic cancer via the miR-338-5p/wif1/wnt8/β-catenin axis.

Similarly, Liu W et al. employed a high-throughput sequencing approach to study miRNA expression profiling in exosomes derived from human UC-MSCs and murine compact bone MSCs (cm-MSCs) to explore their effects on acute graft versus host disease (aGVHD) [121]. Their findings revealed a high expression level of miRNA-223 and identified this miRNA’s mechanistic action in attenuating aGVHD. In another study by Terunuma A et al., the transcriptomes of EV from DP-MSCS and AT-MSCs were analyzed and compared to the transcriptomes of MSCs for the same tissue types [121]. EVs obtained from DP-MSCs exhibit transcriptomic signatures associated with neurogenesis and neural retinal development, while those obtained from AT-MCSs exhibit signatures related to mitochondrial activity and skeletal system development. Notably, the transcriptomes of EV-derived MSCs closely resemble those of their parent MSCs, and genes associated with neurogenesis were found to be highly expressed in both DP-MSCs and their EVs. Conversely, AT-MSCs and their EVs were found to exhibit high expression of genes associated with angiogenesis, hair growth, and dermal matrices. These findings suggest that EVs derived from DP-MSCs may hold promise as a therapeutic target for neurodegenerative disorders and retinal diseases, while EVs-derived AT-MSCs may be useful in rejuvenating the musculoskeletal system and skin.

The transcriptome studies of EVs derived from MSCs have demonstrated the significance of advanced technologies. Specifically, the analysis of the exosome cargo transcriptome can offer valuable insights into the biological activities of exosomes and facilitate the development of effective exosome-based therapeutics. Further investigation into MSC-EVs from diverse tissue types may broaden the range of potential therapeutic targets and enhance our comprehension of the mechanisms underlying the therapeutic effects of MSC-EVs.

4. Strategies to improve the therapeutic outcome of MSCs

While the current clinical successes of MSC therapies are encouraging, reports of failure and minimum efficacy persist. These limitations may not necessarily stem from the potential of MSCs but rather from their suboptimal use. As evidenced by single-cell RNA-seq studies, MSCs are a heterogeneous group of populations with varying cellular characteristics and potential. Additional heterogeneity in the MSC population could be introduced during the manufacturing process (isolation and expansion) [122, 123]. Standardizing the therapeutic potency of MSCs is therefore crucial to ensuring reliable and reproducible results in clinical trials. This can be achieved by establishing consistent protocols for the selection, isolation, and expansion of MSCs, as well as developing protocols for assessing cell quality and potency prior to clinical application. Furthermore, the automation of the expansion process and large-scale production will result in consistent product quality, thereby eliminating variabilities related to the production process. Namely, isolating a homogenous population of cells with the desired potency characteristics and following standardized protocols for expansion can produce high-quality MSCs for therapeutic use.

Another strategy to enhance the therapeutic potential of MSCs is through priming or preconditioning with specific agents before clinical administration. Such a strategy entails priming (treating) MSCs with small molecules, growth factors, and other biological or chemical agents to boost their reparative and regenerative properties and immunomodulatory capabilities. Preconditioning can be viewed as a way of educating MSCs before clinical administration. The main aim is to circumvent problems of MSC proliferation and in vivo survival and to boost the cells’ therapeutic potential. Depending on the intended clinical use of the MSCs, different agents may be used. For instance, the role of growth factors in promoting the survival of transplanted MSCs has been investigated. In cardiac therapy, preconditioning MSCs with TGF-α for 24 hours before transplantation into ischemic sites in a rat model of myocardial infarction increased cell survival [124]. Additionally, priming MSCs with IFNγ was found to enhance their immunomodulatory potential [92]. Alternatively, MCSs can be genetically engineered to induce the expression of genes with desired therapeutic outcomes, including growth factors, cytokines, enzymes, and microRNA, without interfering with cell differentiation and self-regeneration capabilities [125]. Various strategies can be employed for genetic modification to enhance the therapeutic benefits of MSCs while retaining their inherent properties [125].

Cells in vivo reside within three-dimensional (3D) niches, where cell-matrix and cell-cell interactions play a significant role in maintaining cellular characteristics. The current culture protocols for MSCs rely on two-dimensional (2D) expansion systems, which do not accurately recapitulate the native environment of the cells. Consequently, these 2D cultures induce cellular changes that might impact certain therapeutic characteristics of the cells. Differences in the transcriptome profile of MSCs in 2D and 3D cultures have been reported [126, 127, 128]. These differences encompass the expression levels of growth factors and cytokines, which play an essential role in the paracrine mechanism of MSCs’ protection [127, 129]. Therefore, the employment of 3D culture, which closely mimics the native conditions, could be considered an alternative strategy to maintain the therapeutic potential of MSCs. Alternatively, 3D cultures of MSCs could be viewed as a route of cell administration, particularly if they support MSCs’ proliferation and differentiation [130, 131].

5. Conclusions

The clinical relevance of MSCs from various sources cannot be overstated, as they have demonstrated great promise in treating a wide range of diseases and injuries. MSCs are a heterogeneous population of cells with varying inherent potential. Analyzing the whole transcriptome of MSCs offers a robust tool for identifying gene expression patterns and regulatory networks. Through this approach, researchers can gain insights into the molecular mechanisms that govern the properties and functions of MSCs. Identifying key gene expression signatures can help predict the therapeutic potential of MSCs and optimize their use in clinical settings. This high-end technology could help identify the optimal source of MSCs with the desired inherent potential for specific clinical applications. Furthermore, data obtained from analyses could aid in enhancing the efficacy of clinical applications of MSCs. Additionally, new strategies, such as potency testing, priming of MSCs, and the development of 3D biomimicry conditions, should be deployed to enhance the therapeutic outcomes of MSCs.

Acknowledgments

The authors would like to express their gratitude to Professor Abdalla Awidi for his mentorship and guidance.

Conflict of interest

The authors declare no conflict of interest.