Recent advances in understanding mesenchymal stromal cells

Tissue origin of mesenchymal stromal cells

The most prevalent source for MSCs is adult bone marrow¹⁸. Adipose tissue is emerging as an important source, as exemplified by the ATMP granted marketing authorization by the European Medicines Agency (mentioned above). We and others have tried to understand how interchangeable MSCs from different tissue sources are and whether one may be more suitable for certain disease entities than for others. The observed differences suggest an “environmental niche memory”, which could help to select the most appropriate tissue source for a certain clinical indication^12,22–26.

Mesenchymal stromal cell culture conditions and cellular fitness

Usage of fetal bovine serum (FBS) as culture supplement has been a major issue in MSC production²⁷. The growing concern relates to transmission of pathogens such as prions and possible immune reactions against xenogeneic agents^28,29. Consequently, as a replacement of FBS, other supplements have been introduced. One of the most common is platelet lysate (PL)^29–31, as it contains growth factors suitable to support MSC ex vivo expansion without causing genomic instability³². However, the use of PL is not without concerns: batch-to-batch variation and pathogen reduction need to be addressed to standardize PL use in MSC manufacturing³³.

MSC culture conditions differ enormously, hampering comparability of data³⁴. Cellular “fitness” is considered the most critical parameter and is influenced by cellular/replicative age and potential “cryo-injury”^21,35,36.

Expansion of MSCs in vitro, required to achieve clinical doses (see below), ultimately results in replicative senescence that compromises therapeutic efficacy^37,38. Thus, genomic stability should be addressed as a safety measure before clinical application³⁹. In addition, the thawing of cryopreserved MSCs just before transplantation may hamper their therapeutic capacity. In most animal experiments, MSCs are harvested freshly before the transplantation, while on the peak of the replicative phase. Meanwhile, in clinical trial, most MSCs are pre-banked and expanded to their proliferative limit, frozen down, and just thawed prior the transplantation^35,40. Following retrieval from liquid nitrogen, MSCs have been shown to undergo a heat shock response (“cryo stun effect”) leading to cell injury for at least the first 24 hours⁴⁰. This has been shown to compromise immune-modulation function, enhance vulnerability to lysis by immune cells and the complement system, and decrease in vivo persistence upon intravenous administration⁴⁰. A rescue culture for a few days could eventually reduce this “cryo stun effect”.

As it has become clear that culture conditions can greatly affect MSC function, it also opens a new window for MSC priming to improve their therapeutic efficacy. A growing body of data report a wide array of priming approaches, from usage of cytokines, growth factors, hypoxia, pharmaceutical drugs, and 3D culture using biomaterials^41,42. For example, MSC priming with interferon-gamma (IFN-γ) is considered key to suppress T-cell proliferation, partly through production of indolamine-2,3-dioxygenase (IDO) and programmed cell death-1 ligand (PDL-1) upregulation⁴³. Indeed, allogeneic infusion of IFN-γ–primed MSCs to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice reduced GvHD symptoms⁴⁴. However, MSC priming with IFN-γ should be carried out with caution as it can upregulate the expression of HLA class I and II molecules, which could affect immune compatibility⁴⁵.

Lastly, another challenge to bring MSCs to clinical application is upscaling of MSC culture. A number of strategies for upscaling cell, secretome, or extracellular vesicle production have been reported and reviewed extensively^46–48. However, economically feasible approaches that meet Good Manufacturing Practice compliance have yet to be standardized⁴⁹.

Route of application and dosing

Depending on the clinical purposes, MSCs are administered differently, either systemically infused or locally injected. Contrary to the old belief that MSCs migrate to the site of injury and replace the injured tissue once MSCs are injected intravenously, they are mostly trapped in lungs and die within 24 hours⁵⁰. Pulmonary embolism and infarct of three related patients have been reported after adipose MSC infusion⁵¹. MSCs express tissue factor, a cell surface glycoprotein that plays an important role in extrinsic coagulation, which by triggering procoagulation has led to thromboembolic events after MSC infusion. Thus, adding an anti-coagulant during the infusion should be considered⁵².

The majority of preclinical studies using mice and rats infuse around 50 million and 10 to 20 million MSCs per kilogram of body weight, respectively^53,54. Meanwhile, the average number of MSCs transfused intravenously is 100 million per patient, corresponding to 1 to 2 million per kilogram of body weight⁵⁵. This may in part explain the huge discrepancy of outcome between preclinical and clinical studies, assuming that the therapeutic benefit is dose-dependent²¹. Although the notion of increasing MSC dose might be tempting, safety should be assessed carefully for it might, for example, increase the risk for embolism or adverse reactions. In addition, the lack of standardized pharmacodynamics and pharmacokinetics models applied to MSCs represents a limiting factor⁵⁶.

Another potential explanation for the translational gap between clinical and preclinical data is that, in patients, the degree of severity might be too high for MSC therapy to be as efficacious as in animal studies. In order to get better clinical outcomes, MSC-based therapy may be considered as prevention treatment together with first-line therapy and not only as salvage or even palliative therapy. However, this notion will require proper risk–benefit evaluation and support from ethics committees.

Hemocompatibility and immune compatibility

For a long time, MSCs have been considered to be immune-privileged, allowing their transplantation across histocompatibility barriers⁵⁷. Recent data, however, indicate that MSC transplantation may provoke donors’ humoral and cellular immune responses, especially in allogeneic settings^21,58. In GvHD, in fact, this immune recognition appears to be fundamental for the therapeutic effect: MSCs recognized by cytotoxic T cells undergo apoptosis and are phagocytosed by macrophages which subsequently elicit immunosuppression via prostaglandin E₂ (PGE₂) and IDO activities^59–61.

MSCs have also been shown to elicit an instant blood-mediated inflammatory reaction (IBMIR) in both a cell dose- and a donor-dependent manner⁶². Non-bone marrow-derived MSCs appear to express higher levels of pro-coagulant tissue factor, which makes them more likely to induce IMBIR⁶³. Adding anti-coagulants during MSC transplantation may be a good option for clinical application^36,52. Likewise, the selection of tissue factor–negative or low expressing MSCs has been proposed as a strategy to improve hemocompatibility⁶³.

Moreover, allogeneic MSC transplantation can provoke an adaptive immune response in mice through increased T-cell memory and allo-antibodies^64,65. The latter may be associated with complement-mediated cytotoxicity⁵⁸. Yet only two clinical trials^1,67 report the development of donor-specific antibodies in patients who have received allogeneic MSCs⁶⁶. But the authors argue that the increased allo-antibodies have no relevance in the clinical outcome or the occurrence of adverse events^1,67. Moreover, in two other clinical trials, which used allogeneic MSCs to treat type 2 diabetes and diabetic nephropathy, there was no report of patients developing donor-specific HLA antibodies despite donor–recipient mismatching^68,69. Although the results from clinical studies seem encouraging, the scarcity of studies elucidating possible allo-immune reactions may cause a bias in the observed trend⁶⁶. The occurrence of FBS (used as culture supplement for cell expansion)-specific antibodies has prompted the search for alternative and improved culture conditions as described above⁷⁰.

Mesenchymal stromal cell secretome

The MSC secretome is composed of different soluble factors, including cytokines, growth factors, chemokines, immunomodulatory molecules, cell organelles, and nucleic acids, which are produced, some of these eventually encapsulated in extracellular vesicles, and secreted or directly transferred to neighboring cells^71,72. These factors can modulate the immune system, inhibit cell death and fibrosis, stimulate vascularization, and promote tissue remodeling (Figure 2)⁷³. MSCs can adapt efficiently to the local milieu and change their secretome⁷⁴. On one hand, this significantly hampers the understanding of their mechanisms of action (MoAs) in vivo and the establishment of predictive and quantitative potency assays. On the other hand, it paves the way to potentially improve therapeutic efficacy (for example, the preconditioning with different factors that activate very specific signaling pathways). Treating MSCs in vitro with hypoxia, 3D culture, or soluble factors such as stromal cell–derived factor 1 (SDF-1) or transforming growth factor-beta (TGF-β) triggers Akt, ERK, and p38MAPK signaling pathways. These pathways, at the same time, can induce the production of cytoprotective molecules (catalase, heme oxygenase-1, and so on), pro-regenerative (basic fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-1, and so on) and pro-angiogenic (vascular endothelial growth factor, or VEGF) factors, and also immunomodulatory cytokines (IDO, PGE₂, interleukin-6, and so on)^42,71.

Figure 2. Mesenchymal stromal cell (MSC) secretome in clinical application.

Strategies to harness the MSC secretome for clinical purposes include preconditioning/priming of MSCs to manipulate their paracrine factors, isolation of the secretome, and establishing MoA-based potency assay. EV, extracellular vesicle; MoA, mechanism of action.

Extracellular vesicles

Extracellular vesicles (EVs) are further candidates to explain the therapeutic effects of MSCs. EVs are membrane-enclosed particles of different sizes (exosomes, microvesicles, and apoptotic bodies) released by cells in the plasma and other body fluids^75,76. EVs transport biologically active molecules and genetic information to target cells, influencing their function⁷⁷. Thanks to these characteristics, EVs are also emerging as biomarkers for various diseases⁷⁸. EVs carry a wide variety of genetic material, in particular microRNAs, which play an important role in the biological function of EVs. These small RNAs regulate the cell cycle and migration (for example, miR-191, miR-222, miR-21, and let-7a), inflammation (for example, miR-204-5p), and angiogenesis (for example, miR-222 and miR-21). In a new therapeutic approach, MSC-derived EVs are being engineered by increasing or modifying their content (proteins or RNA)⁷⁷. As an example, an effective drug delivery system for wound healing in diabetes was developed by transfecting non-coding RNA (Lnc-RNA-H19) into EVs⁷⁹. Based on these data, some researchers suggest that the conditioned medium or even EVs should be used as drugs rather than MSCs⁸⁰.

In some circumstances (for example, GvHD mentioned above), dead or dying cells may contribute to therapeutic efficacy. Thus, an improved understanding on MSC “necrobiology” has been proposed, considering apoptosis, autophagy, mitochondrial transfer, and also vesicles⁶¹. Recognition by the innate immune system in different disease contexts may be key to understand and improve MSC function^60,81.

Potency assays

For advanced clinical trials, assays that can verify MSC identity and quality and can predict their functionality in vivo are required⁸². Owing to the manifold functions of MSCs and their rapid adaptation to the local milieu, which may modify their function at sites of injury, disease, or inflammation, assays to predict these functions in vivo are hard to develop.

Agreed quality-control criteria include the determination of presence and absence of certain surface markers and of MSC differentiation potential, their senescence status, their secretome and immunomodulatory functions. In addition, surrogate assays which more specifically test the proposed therapeutic mechanism of action, for example angiogenesis have been established^83–87. The group of Galipeau was the first to suggest a combinatorial assay matrix as a platform to integrate different assays^88,89. In the first study, they employed secretome analysis and quantitative RNA-based array to estimate the immunomodulatory capacity of MSCs and their crosstalk with peripheral blood mononuclear cells (PBMCs), in which CXCL9, CXCL10, VEGF, and CCL2 secretion and expression were correlated with suppression of T-cell proliferation⁸⁸. The other study investigated the phosphorylation of signal transducer and activator of transcription (STAT) in MSC-PBMC co-culture settings where STAT1 and STAT3 phosphorylation was associated with MSC immunoinhibitory capacity⁸⁹. Moreover, Phinney et al. reported a “Clinical Indications Prediction Scale” that, based on Twist-1 expression levels, could predict therapeutic efficacy: high levels of Twist-1 predict higher angiogenic potential, whereas low levels are in line with improved anti-inflammatory and immunosuppressive actions⁹⁰.

We expect rapid progress in the development of combinatorial potency assays based on the increasing knowledge of MSC biology (omics, including single-cell analyses)^91,92. Integrating this more comprehensive insight into MSC heterogeneity with MSC molecular signatures and their highly complex interaction with the local microenvironment in line with a better understanding of molecular mechanisms of action in various pathological settings will hopefully enable easy-to-perform assays with predictive value.