Background

Mesenchymal stromal cells (MSCs) are pluripotent non-hematopoietic stem cells with self-renewal capability [1] and being intensively investigated in clinical trials. Since the discovery of MSCs from bone marrow by Friedenstein in 1970s, MSCs have been isolated from various sources including muscle, umbilical cord, liver, placenta, skin, amniotic fluid, synovial membrane, and tooth root [2, 3], and tested in amounts of preclinical and clinical studies (Fig. 1). It is now understood that MSCs have wide-ranging physiological effects including the maintenance of tissue homeostasis and regeneration [4, 5], as well as the immunomodulatory activities suitable for therapeutic application [6]. So their indications have been expanded to graft-versus-host disease (GVHD), multiple sclerosis (MS), Crohn’s disease (CD), amyotrophic lateral sclerosis (ALS), myocardial infarction (MI), and acute respiratory distress syndrome (ARDS) [7,8,9].

Fig.1
figure 1

Various sources of MSCs used in the registered clinical trials. MSCs isolated from bone marrow are most widely applied in clinical trials, followed by those from umbilical cord and adipose. MSCs from muscles, tooth are also used

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Over 300 clinical trials of MSC therapies have been completed in patients including but not limited to degenerative or autoimmune diseases (Table 1 lists some of the representative completed studies). Overall, MSCs have exhibited tolerable safety profile and demonstrated promising therapeutic benefits in some clinical settings, which led to regulatory approvals of MSCs in a few countries. In 2011, the Ministry of Food and Drug Safety (Korea FDA) approved Cartistem®, a MSC product derived from umbilical cord blood and developed by Medipost for the treatment of traumatic or degenerative osteoarthritis [10]. Thereafter, more MSC products including HeartiCellgram®, Mesoblast, TiGenix, and Stempeutics, were approved by regulatory authorities worldwide for the treatment of a variety of diseases. In the USA, Ryoncil (remestemcel-L) is promising to be the first FDA-approved GVHD treatment for children younger than 12, but is still in the stage of safety verification. The amount of clinics offering exogenous stem cell therapies has doubled from 2009 to 2014 in the USA. This boom in stem cell clinics with 351 companies putting stem cells for sale in 570 clinics in 2016 indicated the mal-practice of the MSC therapies [11]. Considering the fact that many of the applied exogenous stem cell therapies lack confirmation on safety and effectiveness from large-scale clinical trials and are even illegal, these medical mal-practices do threaten the development of MSC therapies [12].

Table 1 Some representative registered clinical trials of MSC therapies
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In this review, we will focus on the major challenges of MSC therapies and the underlying factors leading to the failure of clinical trials. Recent advances and prospects concerning the translation of MSC techniques into clinical practices will also be discussed.

Challenges in technology transfer of MSCs from bench to bedside

Although transferring MSCs from bench to bedside is theoretically achievable, substantial failures have been reported in many early- or late-stage clinical trials, which account for the disapproval of many products by FDA [13]. Factors contributing to the failure of MSC clinical development include but not limited to the poor-quality control and inconsistent characteristics of MSCs in terms of immunocompatibility, stability, heterogeneity, differentiation, and migratory capacity [14, 15] (Fig. 2).

Fig. 2
figure 2

The main challenges in clinical applications of MSCs. During preparation of the MSC products, the main challenges include: (1) heterogeneity of MSCs resulted from donor variations such as the health status, genetics, gender, and age. (2) The varying degree of stability of stemness and differentiation capacities between MSCs isolated from different sources, such as bone marrow, adipose tissue, umbilical cord, or muscles. (3) The varying level of expansion capacities under different culture conditions, including confluence, culture surface, oxygen levels, flasks/bioreactors, passage number, and cell surface modifications. At the state of application, challenges remain due to the influence of (1) the homing or migratory capacity of MSCs under different administration route (local/systemic), injection site, infusion time, and cell carrier materials. (2) The immune compatibility between donors and recipients is the key to reduce the risk of rejection, but is affected by environmental inflammatory molecules which could induce distinct expression of MHC-II in MSCs. (3) The complex effective components released by MSCs depending on the host microenvironment (inflammation status, hypoxia, and ECM), which can result in highly variable factors sha** distinct functions of MSCs

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Immunocompatibility of MSCs

MSCs were immune privileged due to the low expression of MHC-I and HLA-I, and no expression of HLA-II or costimulatory factors such as CD40, CD80 and CD86. MSCs can be transplanted as allogeneic cells with a low risk of rejection. Generally, the original MSCs are believed to have low immunogenicity [16]. Most MSC products are manufactured by amplifying a small number of cells obtained from donors, which can increase MSC immunogenicity caused by inappropriate processes and culture conditions. After MSCs infusion, the in vivo inflammatory molecules in turn increase MSC immunogenicity and further decrease MSCs viability and differentiation capacity, particularly when administrating xenogenic MSCs including human MSCs in animal models [17]. Although the primary immunogenicity of MSCs derived from in vitro experiments might be minimal, the secondary immunogenicity induced by in vivo positive feedback loops can cause the absence of efficacy reported in most clinical trials.

Studies have shown that inflammatory molecules (such as interferon-γ), increased cell density, and/or serum deprivation can induce high expression of MHC-II in MSCs, while TGF-β suppresses MHC-II expression [18]. The immune compatibility between donors and recipients is the key to reduce the risk of rejection in the event of long-term treatments with repeated infusions, in conditions requiring promotion of transplanted bone marrow integration, or post-renal transplantation rejection treatments [19]. It has been reported that repeated intra-articular injection of allogeneic MSCs is more likely to cause an adverse reaction than autologous cells when administered in the same manner [20]. The same observations were reported in horses treated with intracellular xenogen-contaminated autologous MSCs (such as FBS) or non-xenogen-contaminated allogeneic MSCs [21].

MSCs of high quality is the first step to ensure the safety and efficacy in clinical trials. Understanding the molecular and cellular mechanisms underlying the immune incompatibility of MSCs will help to improve the manufacture of MSC products.

Stemness stability and differentiation of MSCs

MSCs have mesodermal lineage differentiation potential and the potential to regulate tissue regeneration by mediating tissue and organ repair, as well as replacing damaged cells [Effective components of MSC treatments

The secretion of cytoprotective factors by MSCs was first reported by Gnecchi and colleagues. They observed that Akt-MSCs (MSCs overexpressing Akt) prevented ventricular remodeling and improved the heart function following surgical myocardial infarction (MI). Since cell transplantation and myogenic pathways would be ineffective over such a brief interval, a new mechanism was proposed that the injected MSCs might act through releasing trophic factors that contribute to myocardial protection following an ischemic insult. This hypothesis was then confirmed by evident improvements in cardiac performance following injection of conditioned medium (CM) collected from hypoxic Akt-MSCs into an induced MI model, which protected ventricular cardiomyocytes with less apoptosis when subjected to a hypoxic condition [92].

In 2007, Dai et al. observed that MSCs-CM had a similar, albeit less intense, effect of MSCs in myocardial infarction, indicating that at least part of the effect observed following MSCs injection could be attributed to soluble factors [93]. In the context of neuronal damage, it has been established that the presence of BDNF, GDNF, NGF, and IGF in the MSCs secretome is necessary for the neuronal survival in vitro and in vivo [94, 95]. MSCs-CM has demonstrated therapeutic efficacy in some other disease models including chronic kidney disease, certain lung, and liver diseases [96, 97].

The paracrine effects of MSCs as an initial mechanism of action inspired further biological analysis of MSCs secretome [98]. Subsequent studies found more paracrine effectors, including soluble cytokines, growth factors, hormones, miRNAs, or lncRNAs that targeting a variety of cells such as immune cells and injured tissue cells [99]. In addition, the paracrine effectors could be loaded in extracellular vesicles (EVs) and exerted long-term effects [100]. In accordance, many studies have shown that MSC-derived EVs retain the biological activity of parental MSCs. It has been demonstrated that EVs showed a similar therapeutic effect as MSCs in selected animal models [101]. However, different studies found various effective components of MSCs in specific animal models and human diseases, and the interactions and functional differences between effectors remain elusive. Therefore, novel in-depth analytical techniques and platforms are warranted to investigate the MSCs secretome in the future.

Attempts to improve the therapeutic outcomes of MSCs

Although there were no attributable serious adverse events after MSC therapy, fever within 24 h and temporary pain at the injection sites are commonly occurred. Here we summarize four strategies to limit adverse events related to MSC treatments and improve the therapeutic outcomes, including genetic modifications or priming strategies to change the inherent characteristics of MSCs, and biomaterial strategies to modify the outside circumstances, and the usage of MSCs secretome (Fig. 4).

Fig. 4
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Current attempts to improve MSC treatment. To improve the therapeutic efficiency of MSCs treatment, modification was made mainly in the following aspects: (1) genetic modification of MSCs by viral transduction or CRISPR/Cas9 techniques to engineer MSCs with enhanced homing, potency, or expansion capacities; (2) priming MSCs with small molecules, hypoxia, or structural stimulations by biomaterials to improve MSC function, survival, and therapeutic efficacy, thus boosting their therapeutic efficacy; (3) biomaterial strategies to improve the survival and function of MSCs by offering a scaffold for MSCs adherence, including modifications on dimensionality, stiffness, topographical cues, surface chemistry, and microstructure of biomaterials. (4) Utilize the MSCs secretome as a drug delivery platform for treatment

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Biomaterial strategies to maintain more homogeneous MSCs

Biomaterials for delivering MSCs have been extensively investigated. These materials showed advantages in offering a scaffold for the adherence and survival of MSCs, as well as preserving the functional components MSCs secreted, thus elongating the effective durations in clinical treatment. However, the implantation of biomaterials could induce the foreign-body responses (FBR) in the host immune system, which can potentially result in fibrosis and failure of the implantation. Therefore, biomaterials suitable for MSCs were constructed to ameliorate the FBR and subsequent fibrotic encapsulation [102]. For example, loading MSCs with small-molecule encapsulating microparticles (MPs) can boost the duration of the products. MPs are composed of biocompatible materials that can be therapeutically tuned according to their composition, polymer molecular weight, drug loading, and release capacities [103]. MSCs loaded with degradable budesonide-containing MPs exhibited fourfold increase in IDO activity in vitro compared to MSCs without being pre-treated with budesonide [104]. This led to a twofold improvement in the suppression of peripheral blood mononuclear cells (PBMCs) activation following IFN-γ stimulation [105].

MSCs are typically delivered to a graft site using a decellularized extracellular matrix (ECM) scaffold. The advent of synthetic polymers has revolutionized tissue engineering. These polymers are highly tunable, homogenous, and cell-free materials and have a high batch-to-batch consistency taking the form of porous hydrogels, sponges, plates, or membranes [106, 107]. However, their unique properties could exert different influences on MSCs function. Table 3 summarizes the influence of biomaterials properties on the function of MSCs, including dimensionality, stiffness, topographical cues, surface chemistry, and microstructure of biomaterials.

Table 3 The properties of biomaterials affecting the function of MSCs
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Genetic modification to produce MSCs with desired biologic function

Viral DNA transduction and mRNA/DNA transfection

To further optimize the therapeutic efficacy of MSCs, MSCs have been genetically engineered to produce trophic cytokines or other beneficial gene products in numerous preclinical models by transfecting MSCs with viral or non-viral vectors. Over the last few decades, these MSCs have successfully been engineered to express therapeutic peptides and proteins in animal models [203].

After the disclosure of precise mechanisms of action or key therapeutic factors in MSC-EVs therapy, targeted-EVs could be expanded in uniform proliferative cells such as fibroblasts via gene modification technology. Therefore, with big data-based analysis of transcriptome and proteome, engineered EVs may be manufactured with desired elements. For instance, Thomas C. Roberts et al. engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signaling pathway. Treatment in the Duchenne muscular dystrophy mouse model with these IL6ST decoy receptor EVs resulted in a reduced phosphorylation of STAT3 in muscles; further functional studies verified the in vivo activity of the decoy receptor EVs as a potential therapy [204]. Similarly, CXCR4/TRAIL-enriched exosomes were successfully obtained from MSCs overexpressing both CXCR4 and TRAIL. These exosomes exerted activity as a cooperative agent with carboplatin against brain metastasis of breast cancer in vivo, improving the efficacy of chemotherapy and highlighting a novel synergistic protocol with anticancer agents to treat brain diseases [205, 206]. Moreover, in a Phase 1 clinical trial, IL-12 was engineered to express on the exosome surface using Codiak’s proprietary engEx Platform. This product could enhance the dose control of IL-12 and limit systemic exposure and associated toxicity. EVs can overcome the reported limitations of parental cells on various aspects, including safety, reproducibility, and cost-effectiveness related to storage and maintenance. Engineered EVs might be novel promising therapeutics for clinical application. Furthermore, to resolve current hurdles in EVs-based therapeutics, the production of EVs should be standardized and optimized, and its underlying mechanisms need further investigation.

MSC usage for pandemic diseases such as COVID-19

Pandemic diseases like 2019 novel coronavirus disease (COVID-19) have dramatically increased the number of sickness and death worldwide. Though vaccines have been developed recently, the viruses are still rapidly mutating and expanding, and the available specific and effective treatment options are currently very limited [207]. For severe or critical COVID-19 patients requiring hospitalization, acute lung injures (ALI)/acute respiratory distress syndrome (ARDS) was the main pathologic features, characterized by immunopathological complications with cellular fibromyxoid exudates, extensive pulmonary inflammation, pulmonary edema, and hyaline membrane formation [208]. Besides, inflammation and sepsis are also the leading causes of mortality in COVID-19 patients [209]. In all these cases, any treatment that could hasten recovery would be in substantial demand. MSC therapy may be one such treatment.

MSC therapeutics may be the ideal candidates for handling the broad spectrum of COVID-19 symptoms due to their multifactorial mode-of-action [210]. They can release various factors including keratinocyte growth factor, prostaglandin E2, granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-13 to facilitate the phagocytosis and alternative activation of alveolar macrophages, alter the cytokine secretion profile of dendritic cell subsets, and decrease the release of interferon γ from natural killer cells [211]. For example, IL-10, TGF-β, and tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase secreted from them were reported to suppress the proliferation of T cells and change the cytokine secretion profile of T cell subsets [212]. Moreover, the proliferation, differentiation, and chemotactic properties of B cells were impaired by MSCs as well. Except for the immune regulatory effects, MSCs can enhance the restoration of capillary barriers, inhibit bacterial growth, and restore alveolar ATP. All these functions mentioned above might also be effective in COVID-19 infection.

COVID-19 has been the top priority of global healthcare systems since its emergence. There have been more than 160 vaccines in development and more than 60 clinical trials are ongoing, and now, only a few vaccines have been approved [213]. The representative clinical trials of MSC therapy in COVID-19 disease were listed in Table 8. But the rapid mutation of SARS-CoV-2 virus leads to challenges on the effect of the available vaccine. It is an urgent need to develop more universal and stable therapy to reverse or combat. Though no evidence has showed that coronavirus was eliminated completely after stem cell treatments, preliminary results were promising. Diseased patients were more likely to survive the infection after the treatment. The specific primed MSCs were also investigated for COVID-19 treatment [212, 214]. The results will provide a strong foundation for future scientific research and clinical applications for a variety of diseases including pandemic crisis and pulmonary complications. Hopefully, the approaches utilizing MSCs particularly the primed MSCs could be vital for the success of cell therapy in treating COVID-19.

Table 8 MSCs therapies for COVID-19 in clinical trials
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Conclusions

Although MSCs therapies have achieved tremendous advancements over the past decades, substantial challenges remain to be overcome. The main challenges include the immunocompatibility, stability, heterogeneity, differentiation, and migratory capacity. More and more studies are focusing on the attempts to overcome these shortcomings. Although the detailed mechanism of MSCs immunomodulatory effects is still elusive and any attempts to improve MSCs efficacy are still lack of evidence, the preclinical studies are develo** rapidly and more standardized clinical trials are wildly carried out. It might be expected that the conversion to canonically registered MSC therapies will flourish with time. The lessons from the current MSCs investigations may provide critical guidance for investigators pursuing further translational processes. With the clarification of MSCs effectors and the emergences of new technologies assisting in-depth studies, MSCs are promising to be proved as effective treatment options for a variety of devastating conditions.