Introduction

Cell therapy has become one of the most important emerging medical treatments in the world. Treatments utilizing stem cells, induced pluripotent stem cells (iPSCs), somatic cells, and immune cells are well documented [1]. Many cell therapy products have already received global market approval. Among them, the mesenchymal/stromal stem cells (MSCs) present a promising tool for the treatment of various diseases.

MSCs were first isolated and described by Friedenstein and his colleagues as adherent and highly replicative cells that can differentiate into mesodermal lineages including osteoblasts, chondrocytes, adipocytes, and hematopoietic stroma [2]. Since then, these cells have gained attention in the field of cell therapy for their tropism towards injured/inflamed tissues, their immunomodulatory capabilities [3], and their relative ease of isolation and expansion [4]. MSCs can be isolated from many sources, including bone marrow [5], umbilical cord [6], adipose tissue [7], cord blood [6], placenta [8], dental pulp [9], endometrium [10], amniotic fluid [11], skeletal muscle tissue [12], lung tissue [13], liver tissue [7, 12] and dermal tissue [12], and many of these cells have been used in clinical studies (Fig. 1a). The characteristics of MSCs make them attractive as cellular therapeutic agents for regenerative medicine and immune-related diseases.

Fig. 1
figure 1

MSC sources and clinical indications in clinical studies. As of October 11, 2020, 1,242 registered studies were identified on clinicaltrials.gov by searching keywords “mesenchymal stem cell” or “mesenchymal stromal cell” (Additional file 1). After excluding studies with no longer available/ suspended/ temporarily not available/ terminated/ unknown/ withdrawn status, unknown phase information, and studies that did not use MSCs in their intervention arm, 639 studies remained. Nine of these 639 studies investigated MSCs from two tissue origins, generating a total of 648 studies for analysis. a Tissue origins of MSCs in clinical studies, b number of MSC-related clinical studies by medical specialty, and c the top 20 disease indications of MSC-related clinical studies

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The first clinical trial of MSCs was reported in 1995 in patients with hematologic malignancies. Lazarus et al. demonstrated that ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (BMMSCs) in patients caused no severe adverse effects [14]. Subsequently, treatment with BMMSCs was shown to provide clinical improvement in the rare skeletal disease osteogenesis imperfecta [15]. Furthermore, many clinical trials have examined the feasibility and efficacy of MSCs for the treatment of various conditions, including acute organ failure [16,17,18], graft-versus-host disease (GVHD) [19,20,21], ischemic heart disease [22, 23], cardiovascular disease [24, 25], liver cirrhosis [26], diabetes [27, 28], spinal cord injury [29,30,31], and bone/cartilage injury [32,33,34,35,36,37] (Table 1). According to the National Institutes of Health (http://www.clinicaltrial.gov/), the number of registered MSC-based clinical trials was over 1,200 as of October 11, 2020, of which approximately 600 had defined phase and status (Fig. 1b, c, Additional file 1 and Additional file 2). Most of the studies to date are phase 1 and phase 2 trials which evaluate safety and feasibility, and evidence of therapeutic efficacy is still lacking (Fig. 1). The most common indications of MSC-based cellular therapy include osteoarthritis, ischemic heart disease, graft-versus-host disease, spinal cord injury, and multiple sclerosis (Fig. 1c). In addition, since the elevation of coronavirus disease-19 (COVID-19) outbreak to pandemic status on March 11, 2020 [38], numerous MSC-based studies have been registered, and COVID-19 related pneumonia and acute respiratory distress syndrome (ARDS) has risen as the second most common indication as of October 11, 2020 (Fig. 1c). The rapid global response and increase of COVID-19 related MSC trials highlighted the promise of MSCs in treatment of inflammatory and immune diseases.

Table 1 Summary of MSC-based clinical/preclinical trials
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Although studies on MSCs are well-documented, MSC-based cellular products still have not been approved by the US Food and Drug Administration. The lack of consistent and standardized methods for characterizing the safety and efficacy of MSC products is a major concern, which dramatically slows the progress of MSC therapy towards clinical use. The safety of cellular products is always the first priority. Although some MSCs have been shown to be safe for clinical use in a previous meta-analysis, whether this conclusion can be extended to MSCs from other tissue origins or different culture conditions is still uncertain (Fig. 1a) [39]. The risk associated with MSC products centers around their capability to initiate and promote tumors. These risks, as well as the biodistribution of systemically administered cells must be better clarified before the widespread use of MSCs in clinical practice. In this review article, we focus on the effects of MSCs on tumor promotion and suppression, and discuss methods to study their biodistribution.

MSC-based mechanisms of action

Several possible mechanisms by which MSCs exert their beneficial effects have been proposed. Early studies reported that MSCs could migrate to sites of injury and then differentiate into functional cells [40], or that they could fuse with compromised cells to regenerate damaged tissues [41, 42]. More recent studies have demonstrated that paracrine factors [43, 44], mitochondrial transfer [45], and extracellular vesicle secretion [46] have important roles in mediating the effects of MSCs.

Paracrine effects

MSCs secrete paracrine factors, including cytokines, chemokines, growth factors, and miRNAs. MSC transplantation or administration of isolated secreted factors enables MSC paracrine factors to get to injured tissues, to help restore a healthy microenvironment to promote tissue repair [47] (Table 2). MSC paracrine factors play important roles in immunomodulation [48, 49], tissue regeneration and healing [50, 51], anti-fibrosis [52, 265], as pcMSCs are more than ~ 20 μm in diameter and much larger than the width of the micro-capillaries of the lung. After intravenous infusion, FND-labelled pcMSCs disappeared from the lungs as time passed, and migrated to other tissues/organs such as the liver and spleen, or to injured sites. Nevertheless, the number of FND-labelled pcMSCs decreased in the heart and kidneys (Fig. 5b).

As it has been reported that MSCs will migrate to injured sites [266], we induced an ischemia–reperfusion injury to the left kidney in our animal model (Fig. 6a) and examined whether FND-labelled pcMSCs injected into the portal vein would appear in the injured kidney, to test the concept that MSCs will migrate to sites of injury. In our mouse model with healthy kidneys, the number of pcMSCs in the kidneys decreased over time (Fig. 6b, upper panel) and the decrease was evident in both the left and right kidneys. (Fig. 6b, lower panel). In contrast, in the mouse model with the injury the number of FND-labelled pcMSCs in the injured kidney was highest on day 5 (3%; Fig. 6c). As seen in the lower panel of Fig. 6c, the injured kidney (L kidney) had significantly more FND-labelled pcMSCs than the healthy kidney (R kidney). The percent of FND-labelled pcMSCs remained consistent over time (~ 0.25%) in the healthy right kidney (R kidney) (*P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001.) (Fig. 6c, lower panel). Given these data, it appears that the percentage of MSCs that migrate to kidneys is limited to about 4%, and it appears that the kidneys have the ability to redistribute MSCs in vivo. In addition to providing fast and accurate results, this technique is completely safe to the cell tissue. The FND-labelling technique does not alter any properties of the cell, including cell viability, proliferation, differentiation and immunomodulation, making this method very biocompatible.

Fig. 6
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Fluorescent nanodimond (FND)-labelled pcMSC biodistribution analysis in mouse model with a kidney ischemia–reperfusion injury. a Timeline of the ischemia–reperfusion kidney injury mouse model. The ischemia–reperfusion injury was created on the left-hand side kidney (L) in a mouse, then FND-labelled pcMSCs were injected through the portal vein. b Bodistribution of FND-labelled pcMSCs in healthy kidney mouse model. Experiments were repeated in triplicate and error bars represent the standard deviation of the measurements. c Biodistribution of FND-labelled pcMSCs in ischemia–reperfusion kidney injury mouse model. Experiments were repeated in triplicate and error bars represent the standard deviation of uncertainty. Data are presented as mean ± standard deviation. Data were analyzed using Student’s t-test. *P value of < 0.05. **P value of < 0.01. ***P value of < 0.001. ****P value of < 0.0001

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Clinical applications of MSCs in cell therapy: safety and potency

The potential and promise of MSC therapy is highly anticipated in recent and coming decades. As with all emerging new medical technologies, patient safety is always the first priority. As we have discussed, although the ability to modulate immune environment and promote tissue regeneration have been well reported in preclinical studies, the aspect regarding tumor induction or promotion is still one of the many concerns. The MSCs derived from different tissue origins or expanded under different culture conditions present different immune profiles which may result in tumor promotion [126]. Additionally, as the double sided blades of the MSCs’ strong immune modulation ability [262], evaluation of both the specific MSC properties as well as the patient’s immune conditions is strongly needed. The patient’s immune condition both before, during, and after treatment should be closely monitored.

Some reports showed that artificial engineering process may decrease the tumor induction and increase tumor-suppressing function of MSCs [263]. However, genetically engineered MSCs also raise other safety concerns. Although several clinical trials claimed the safety of MSC-treated patients, however, most of the trials only showed short-term safety and are without the examination of tumor-associated biomarkers [267, 268].

A recent systematic review and meta-analysis reappraised 55 randomized controlled trials and over 2000 patients to investigate the safety of systemically inoculated MSCs [39]. The risk of fever was significantly greater in the group of patients receiving MSCs. There was no significant increase in the risk of infection, thrombo-embolic events, malignancy or ectopic tissue formation, while the risk of death was significantly lower in the MSC-treated patients. Among the included studies, severe adverse events, including treatment related fever, in-stent thrombosis with death, acute coronary artery occlusions after intra-coronary delivery, grade 1 anaphlyactoid reaction, gastric ulcer perforation, hypersensitivity reaction, and anal cancer, have been reported to be possibly related to MSC treatment. Although the conclusion of the meta-analysis ends on a promising note, it was also emphasized that an a priori plan to monitor safety should be outlined in every clinical study design, including immediate allergic reactions, local complications (hematoma formation, local infection), vascular obstructions (dyspnea, oliguria, myocardial infarction, venous thromboembolic events), systemic complications (systemic infection, abnormal liver or renal function), malignancy or ectopic manifestation of implanted MSCs, and other disease-specific safety considerations [39].

Additionally, patients with medical history of ischemic diseases, cardiovascular diseases, lung fibrosis, concurrent neoplasm, and family history of hereditary cancer should be carefully reviewed during MSC treatment. The cell dose, infusion route and rate should be documented. The product profiles of the MSCs from different tissues and different generation processes, such as transcriptome, epigenome, proteomic data, cell populations, potential potency biomarkers, preclinical data from cell and animal studies, should be provided.

The therapeutic efficacy of MSCs in different disease indication is still under evaluation, as most of the studies to date have been limited to phase 1 and phase 2 studies (Fig. 1b and 1c, Additional file 2). As we have discussed in this review, the differences in MSC tissue origins and the variety of cell culture conditions would be some of the important factors determining MSC potency in vivo [269]. Thus, the development of surrogate potency assays using preclinical animal model is needed [270]. Recently the International Society for Cellular Therapy (ISCT) have announced some strategies to identify the potential effective factors of MSC action mechanism, including the combined the matrix assay and multiple techniques, such as quantitative RNA analysis for the specific genes, flow cytometry analysis for cell surface markers, and the protein-based assay of secretome [271]. Potency assessments in evaluating cell pharmacology, cell delivery route, as well as the cell-drug interaction are still under development to improve the MSC precision therapy [272,273,274,275]. Although the matrix assays were reported to serve as a platform to identify the biomarkers for MSC potency in vitro [276, 277], whether this in vitro assays are able to identify the MSC potency are still under discussion. For example, the use of allogeneic human peripheral blood mononuclear cells for mixed lymphocyte reaction (MLR) assays is a popular assay to demonstrate the MSC immunomodulation capacity. However, the lack of robustness, accuracy, and reproducibility is of concern [278,279,280]. Additionally, the correlation between the in vitro assays and in vivo pre-clinical/clinical data requires further evaluation.

Cryopreservation could be another factor affecting MSC potency. It has been documented that the MSC cryostorge, the so-called “cryo stun effect”, may decrease MSC therapeutic efficacy, leading to failures in MSC clinical trials [278]. Recently, a systematic review regarding the impact of cryopreservation on BMMSCs showed that the cryopreservation appears to affect the cell viability, apoptosis, cellular attachment, immunomodulation, and metabolism of BMMSCs [279]. Furthermore, these impaired viability or functions of the MSCs can be restored, partially or totally, by following an acclimation period [279,280,281], or by IFNγ licensing before cryopreservation [282].

In summary, the use of standardized potency assays should be incorporated into future MSC product release criteria. Thus, development of surrogate potency assays for different disease indications should be highlighted. The optimal process of cryopreservation and thawing may be another important factor requiring further attention.

Conclusions

MSCs are a major cornerstone to the advancement of cell therapy, yet much remains to be learned about their pharmacokinetics and pharmacodynamics after systemic application in vivo. The different tissue origins of MSCs not only confer different biological activities that affect their therapeutic usefulness, but also raise the concern of different safety profiles. Many methods, including herein discussed fluorescent nanodiamond, are available for tracking inoculated MSCs in vivo, each with different advantages and disadvantages. These imaging platforms will facilitate future studies to discern and optimize the use of different MSCs for future clinical therapies.