Over the past several decades, incidences of damage, failure, dysfunction of multiple tissues and organs have reached epidemic proportions (1–3). However, scarcity of donors, limited availability of autologous tissues, risk of chronic tissue rejection, lack of a cost-effective therapeutic strategy, and the overall financial burden of caring for affected individuals present challenges in developing advanced therapeutic options (4–9).

The advent of regenerative medicine has provided immense hope to the primary researcher and clinician to use stem cells with a determined aim of rejuvenating the damaged tissues and organs (10–12). Numerous animal models have been used to test stem cell types (e.g., embryonic, induced, and adult) over the past years for autologous and allogeneic therapies (13–16). Although having a relatively limited differentiation potential in comparison to the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the combination of factors like autologous cell therapy, reduced possibility of teratoma formation, immunological intolerance, xenogeneic contaminations, lack of ethical concern, and the reduced financial burden have made mesenchymal stem cells (MSCs), an ideal choice for the regenerative cell therapy (17–21). Despite the promising therapeutic potential of MSCs in regenerative medicine, few of the critical questions, such as what are the precise molecular mechanisms responsible for providing the host's immune tolerance against MSCs, is the clinical application of MSCs safe, and how can we increase the scalability and yield per batch, remain unanswered and have imposed significant challenges in their clinical translation, prompting researchers to explore alternative tissue regeneration approaches.

Recent studies propose that MSCs profoundly manifest their action by the paracrine secretion of extracellular vesicles (EVs), particularly exosomes (22–25). The unique potential of these MSCs-derived exosomes (MSCs-Exos) to recapitulate the stem cell properties have paved the path for a “cell-free” therapy in the field of regenerative medicine. Furthermore, in recent years, it has been substantially demonstrated that MSCs-Exos cargo is enriched in distinct ncRNAs, specially-microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and Piwi-interacting RNA (piRNAs), mediating tissue communication, modifying cellular phenotype, regulating wide range of biological, pathophysiological processes making them a relevant drug delivery and therapeutic option in the field of regenerative medicine (26–30).

In the current review article, we aim to summarize the emerging role of MSCs-Exos-derived ncRNAs (MSCs-Exos ncRNAs) in regenerative medicine and diseases. We further emphasize the regulatory mechanisms, therapeutic approaches, ongoing clinical applications, and the challenges associated with MSCs-Exos ncRNAs in the field of regenerative medicine and tissue engineering.

MSCs are stromal, nonhematopoietic cells generally obtained from different sources including bone marrow, adipose tissue, umbilical cord, placenta, amniotic fluid, and dental pulp (31–37). The characterization of the MSCs depends upon the expression of specific cell surface markers (CD29, CD37, CD73, CD90, CD102, CD105, and CD166) and their potential to differentiate into multiple cell types such as osteocytes, chondrocytes, and adipocytes under appropriate, stimulating culture conditions (38–43). Recent clinical and preclinical trials have demonstrated the potential of MSCs to be an excellent autologous cell source treating numerous diseases including diabetes, myocardial infarction, osteoarthritis, Alzheimer's, both in animal models and patients (44–49). Although MSCs-based therapy has proven beneficial in treating a wide range of diseases, we still have incomplete knowledge regarding their mechanism of action. Emerging studies in recent years have found that MSCs mediate their action via the paracrine secretion of exosomes (Figure 1), regulating numerous biological processes (50).

Exosomes are membrane-enclosed spherical or cup shaped endocytic vesicles with a size ranging from 40 to 150 nm formed intracellularly in the cellular multivesicular bodies and released from numerous cell types (51–53).

Exosome biogenesis occurs via three pathways: ESCRT-dependent, ESCRT independent, or direct budding of the plasma membrane. In the first two pathways, an early endosome is formed by the inward budding of the endosomal membrane to generate intraluminal vesicles (ILVs). These ILVs accumulate to form multivesicular bodies (MVBs), which fuse with the plasma membrane (facilitated by Rab GTPases). The ILVs are then released from cells as exosomes, where they are taken up by recipient cells via endocytosis, direct binding, or ligand-receptor binding. The Endosomal Sorting Complexes Required for Transport (ESCRT) includes four distinct proteins: ESCRT 0, I, II, and III. Briefly, ESCRT 0 relegates ubiquitinated proteins within the endosomal membrane and recruits ESCRT I and II. ESCRT I and II bind near ubiquitinated proteins on the outer surface of the endosomal membrane, “tagging” them for recruitment within newly forming intraluminal vesicles in the MVB. ESCRT III then sequesters MVB proteins, finalizing the process of exosome formation. Exosome biogenesis via the ESCRT-independent pathway was discovered recently after knock-out studies involving the ESCRT complex proteins showed that the cells could continue the process of exosome formation and release. This pathway involves lipids such as sphingolipids and ceramides and proteins such as heat shock proteins and tetraspanins (54–56). Why these distinct pathways exist, and which pathway is used in cells is still poorly understood. Investigators have used different approaches to increase the angiogenic potential of exosomes released by the stem cells (57). The release of exosome can be considerably increased in vitro using stress conditions that mimic organ injuries, such as hypoxia, irradiation, or drug treatments.

The MSC-Exos have been tested widely on human patients of ischemic heart injury, cerebrovascular disease, and liver fibrosis as an alternative to MSCs themselves (58–61). Intriguingly, numerous advantages over MSCs such as increased viability, higher uptake, lower immune response, reduced risk of embolism, and potential to cross the blood-brain barrier have made MSC-Exos a promising candidate emerging as an effective “cell-free” therapeutic approach in the field of regenerative medicine (22, 62–64).

In recent years, considerable amounts of research depict MSCs-Exos ability to facilitate the exchange of genetic and epigenetic information intercellularly leading to the modulation of neighboring and distant cells' gene expression in both healthy and disease states (65). Preclinical data further demonstrate that the acquired repertoire of biological cargo, primarily ncRNAs of MSC-Exos, has been found to effectively disseminate biological information between cells and regulate the inflammatory apoptotic pathways in various and disease states (64, 66).

In the next section, we represent the overview of ncRNAs from the MSC-Exos in context of regenerative medicine. We also discuss their role as diagnostic and therapeutic targets for regenerative medicine and tissue engineering.

In the past two decades, advancements in high throughput sequencing technologies and computational approaches have successfully captured and annotated the transcriptome revealing the non-coding transcripts and their functionality in the human genome (67, 68). The extensive information from these in-depth analyses demonstrates the genome complexity, with <2% of the genome encoding than a large percentage (98%) being transcribed into heterogeneous ncRNAs transcripts (69).

The ncRNAs comprises of a diversified repertoire of endogenous RNA transcripts including short and long (<200 nucleotides) ncRNAs with no protein-coding potential (70, 71). Initially, the ncRNAs were considered as transcriptional junk having no biological function; however, in recent years, numerous studies have depicted the regulatory function of ncRNAs modulating the expression of genes involved in critical biological processes (72). Moreover, the dysregulated expression signatures of ncRNAs are found to be contributing toward the pathogenesis of various diseases by diverse mechanisms (Figure 2) (73–77).

The MSCs are active sources of exosomes with enriched cargo, including lipids, DNA, RNAs (mRNAs, miRNAs, lncRNAs, circRNAs, and piRNAs) proteins. MSC-Exos are also being explored as viable biomarker in diseases. Their cargo of non-coding RNA (ncRNA), including lncRNA and miRNA, is being studied for their role as potential therapeutic strategies in regenerative medicine (Table 1).

Neurodegeneration is a persistent barrier to the effective recovery of patients following a cerebrovascular accident (CVA) with ischemic stroke (IS). According to the American Heart Association, IS has become the fifth leading cause of death in the United States (125). A blockage in the cerebral arteries characterizes ischemic stroke, predominantly occurring in the middle cerebral artery (MCA), which subsequently prevents blood flow to the brain. Thrombolytic therapies are the first-line interventions administered following an ischemic stroke, but their effectiveness decreases as the time from stroke to hospital admission increases (126). Lack of oxygen following blockage of the cerebral artery causes downstream cerebral tissue hypoxia and cell death, leading to irreversible damage to brain tissue and accompanying neurocognitive disabilities (127). Researchers have been working to regenerate injured brain tissue following IS using mesenchymal stem cell-derived exosomes as increasing evidence has pointed to the therapeutic role of miRNAs in attenuating cell damage and inflammation following brain injury (128–131).

A primary goal in treating ischemic stroke is decreasing compensatory injury to nerve cells deprived of oxygen downstream of the vascular blockage. Rapid reperfusion is performed to reinstate oxygen to the affected tissue to rescue cells from hypoxia-induced damage, though only a limited time window is allotted for effective reperfusion. Various miRNAs are involved in the mitigation of ischemic stroke, whereby their exogenous introduction to tissues via exosomes has been shown to remediate damages associated with CVAs. Xin et al. (95) observed that MCAO/R rats exhibited improved functional outcomes and increased neurite remodeling in the ischemic boundary zone (IBZ) following administration of miR-133b-enriched MSC exosomes.

Inflammatory processes in hypoxic tissue pose another hurdle to effective recovery of patients following IS as persistent activation of inflammatory mediators results in tissue edema and prolonged recruitment of leukocytes to the affected area (132). Cytokines released from injured neural tissue activate inflammatory pathways, of importance being the NF-kB pathway, known for producing proinflammatory cytokines such as IL-1, IL-2, IL-12, and TNF-α, among others. Toll-like receptor 4 (TLR4) is a PRR located on the cell surface of microglia and other macrophages and is involved in intracellular signaling via the NF-kB pathway (133). Cai et al. (96) showed that decreasing the expression of TLR4 via delivery of miR-542-3p enriched MSC-exosomes to MCAO/R mice reduced infarct volume, edema, and infiltration of inflammatory cells.

Augmenting the inflammatory response through controlling microglial polarization is an attractive route for mitigating cerebral injury following IS. Microglia are macrophages that are key players during the inflammatory process during and after IS and are responsible for both innate and adaptive immunity in the brain. Their polarization into either the M1 or M2 phenotype is characterized by pro-inflammatory or anti-inflammatory cytokine production, respectively (134). Zhao et al. were able to induce M1 to M2 microglial polarization in BV2 mouse microglial cells via the introduction of miR-223-3p-enriched MSC-exosomes (ex miR-223-3p). miR-223-3p was found to decrease cysteinyl leukotriene receptor 2 (CysLT2R), which may play a role in the induction of the M1 macrophage phenotype. Treatment with ex miR-223-3p on BV2 cells induced their conversion from the M1 to the M2 phenotype while in vivo delivery of ex miR-223-3p decreased production of pro-inflammatory cytokines (IL-1β and IL-6) while increasing the production of the anti-inflammatory cytokine IL-10 and reduced infarct volume in conjunction with improving the functional recovery of rats, including activity, learning, and memory (97).

Neurodegeneration remains an imminent barrier to the recovery of patients following IS. Exosome-derived non-coding RNAs serve many biological processes and are critical mediators in the regeneration and protection of nerve tissue following IS-induced brain injury.

MSC-derived exosomes are currently being explored as viable biomarkers in disease and their cargo of non-coding RNA (ncRNA), including lncRNA and miRNA, are being studied for their role as potent therapeutic strategies in regenerative medicine. This RNA cargo is also being explored in the context of cancer therapeutics while the exosomal skeleton is being looked at as a possible vehicle for delivery of chemotherapeutic agents. The role of MSC-derived exosomes in cancer is quite a contested subject, as numerous reports have provided evidence that various MSC-exosomal lncRNA and miRNA can either inhibit cancer cell proliferation/induce apoptosis or induce metastasis/cancer progression (135–140).

In the recent years, KRAS targeting has emerged to be an efficient therapeutic efficacy in context to lung and pancreatic cancer as KRAS mutations are found in 30% of lung and 90% of pancreatic carcinomas. Yet, KRAS has been denoted an “undruggable” target (in part due to its high affinity for GTP/GDP, smooth surface with limited binding pockets, lack of allosteric binding sites, and extensive post-transcriptional modifications. H3K27me3 methylation is associated with the downregulation of nearby genes, of importance being K-Ras (involved in the Ras/MAPK pathway) (141–143). Xu et al. (135) demonstrated that miR-124 from bone marrow-derived mesenchymal stem cells (BM-MSCs) directly inhibited expression of EZH2, which suppressed proliferation, migration, and epithelial mesenchymal transmission (EMT) while inducing apoptosis in pancreatic cancer cells (Aspc-1 and PANC-1).

Xu and colleagues found that MSC-derived exosomal miR-133b inhibited proliferation, migration, and invasion of U87 glioma cells by decreasing EZH2 expression in vitro and inhibited tumor growth in vivo via modulating the Wnt/β-catenin signaling pathway by inhibiting the expression of EZH2 (136). In another study, miR-133b was shown to suppress glioblastoma invasion and cell migration via downregulation of metalloproteinase 14 (MMP14) in U87 and U251 glioma cells, while miR-133b was also shown to impede proliferation and invasion of glioma due to Sirt1 downregulation (144, 145).

Pakravan et al. found that MSC-derived miR-100 suppressed angiogenesis in breast cancer cells by altering the mTOR/HIF-1α/VEGF signaling axis. mTOR is a protein kinase involved in the PI3K/AKT pathway and is known to drive HIF-1α accumulation, which acts as a transcription factor by binding to promoter regions in genes known as hypoxia-response elements (HREs). HIF-1α binds to the HRE located within the vascular endothelial growth factor (VEGF) promoter to increase expression of VEGF and drive angiogenesis. miR-100 derived from MSCs was shown to decrease expression and protein levels of mTOR, HIF-1α, and VEGF in MDA-MB-231 and MCF-7 breast cancer cell lines (known to overexpress VEGF) when co-cultured with MSCs containing miR-100 (146).

An opposing conclusion was made regarding the therapeutic use of MSC-derived exosomal ncRNA and cancer, whereby expression of various lncRNAs (AGAP2-AS1 and HCP5) promoted stemness and drug resistance in breast cancer and gastric cancer, respectively. Han et al. (140) found that AGAP2-AS1 lncRNA was upregulated in trastuzumab-resistant breast cancer cells co-cultured with MSCs harboring lncRNA AGAP2-AS1. AGAP2-AS1 was shown to interact directly with HuR (known to stabilize CPT1 mRNA) and increase fatty acid oxidation (FAO) to promote stemness and drug resistance. Wu et al. found that MSC-derived HCP5 lncRNA increased FAO in gastric cancer cell lines via sponging to miR-3619-5p (found to inhibit expression of CPT1) by affecting the AMPK pathway via PPARGC1A and PGC1α, leading to stemness and chemo-resistance. Overexpression of miR-3619-5p significantly decreased expression of PPARGC1A, which decreased PGC1α (product of AMPK pathway), a transcriptional co-activator of FAO genes (137).

The regulation of cancer progression and inhibition via modulation of oncogenic pathways using MCS-derived ncRNA is an area that must be studied in greater depth. A solid understanding of ncRNA targets is needed to come to a consensus about the potential use, safety, and benefits of using exosome-derived ncRNAs in cancer therapy.

In the retina, the exosomes derived from human U-MSCs (huc-MSCs) expressing miR-126 demonstrated remarkable therapeutic effects by decreasing hyperglycemia associated with retinal inflammation by downregulating the high-mobility group box 1 expression (HMGB1) in diabetic rats and human retinal endothelial cells (HREC) (147). In a murine subretinal fibrosis model, huc-MSCs exosomes containing miR-27b-3p could mitigate subretinal fibrosis by reversing the process of epithelial-mesenchymal transition (EMT) induced by TGF-β2 via inhibiting homeobox protein Hox-C6 (HOXC6) (148). The exosomal lncRNA H19 contributed to wound healing by preventing the apoptosis and inflammation of fibroblasts by impairing miR-152-3p-mediated PTEN inhibition in diabetes and foot ulcer disease (DFU) (149). A study of huc-MSCs in an experimental rat model of acute lung injury (ALI) revealed that exosomal miR-22-3p suppressed pathological changes, apoptosis, and NF-κB expression in LPS-treated rats (150).

In summary, MSCs improve the regenerative potential in the above-mentioned diseases by transporting the exosomes enriched ncRNAs having anti-apoptotic, and anti-inflammatory effect.

The MSCs exosomes impart the regenerative effect on the target cells via ncRNAs, possibly making them a promising candidate in the tissue engineering field. However, the mechanisms associated with the ncRNAs packaging, uptake, release, and the underlying regulatory mechanisms are still in their infancy.

Many autoimmune diseases are characterized by an activated inflammatory state resulting in infiltration of the autoreactive immune cells damaging the tissues. Interestingly, MSCs-Exos in many autoimmune diseases have successfully induced immune polarization from an inflammatory to an anti-inflammatory state, mitigating the disease pathogenesis. Much of this might be mediated by the ncRNA repertoire in the exosomes. So, first, it's essential to determine the driving mechanism behind packaging the altered ncRNAs into the exosomes under a specific disease state. Secondly, it would be important to decipher the feedback mechanisms by which specific immune cells can control the exosome release as priming the MSCs to release the exosome more efficiently and enhance the therapeutic efficacy under disease conditions.

In addition to the challenges mentioned above, the low yield of exosomes/MSCs, their purification, storage, optimized dose to be administered would need further development. Therefore, the clinical application must scale up the production of MSCs-Exos without compromising their purity and stability. The concerns raised above impose significant challenges before bringing MSCs-Exos into clinical practice. Nevertheless, working on few of these domains might improve their regenerative potential.

The potential of the exosomes to recapitulate the therapeutic properties of the stem cells has paved the path toward cell-free alternative therapy, allowing researchers to use them in a multiple diseases and disorders. The underlying factors and the associated mechanisms contributing to the exosomes' regenerative effects have been an area of interest in the past few years. The recent studies depict that the regenerative effects of the exosomes can be ascribed to their ncRNA repertoire. Moreover, modulating the expression of the ncRNAs in the exosomes has allowed researchers to extensively evaluate the protective effects in tissue repair and regenerative medicine. It will be interesting to investigate how the modification in the intracellular and extracellular conditions of the MSCs itself would change the ncRNA repertoire. In addition to that, it would be important to investigate if these changes could eventually generate exosomes having a homogenous expression of the ncRNAs and could be used safely in the clinics.

TP and AD conceived the original idea. TP and MJ collected and prepared the literature, reviewed the literature, and wrote the original manuscript. TP, AD, MJ, and ZB contributed to interpretation of the literature. All authors read, discussed, and revised the initial manuscript and read, approved, and contributed to the final manuscript.

This work was supported in part by DST-UKEIRI DST/INT/UK/P-50/2012 to AD and TP and by the grant P01GM066730 from the National Institutes of Health, Bethesda, MD, to ZB and TP. The funding body had no role in the design of the study, collection, analysis, interpretation of data, and in writing the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.