s41551-018-0325-8

Review ARticle

https://doi.org/10.1038/s41551-018-0325-8

Manufacturing of primed mesenchymal stromal cells for therapy

James Q. Yin  1*, Jun Zhu1 and James A. Ankrum  2*

Mesenchymal stromal cells (MSCs) for basic research and clinical applications are manufactured and developed as unique cell products by many different manufacturers and laboratories, often under different conditions. The lack of standardization of MSC identity has limited consensus around which MSC properties are relevant for specific outcomes. In this Review, we examine how the choice of media, cell source, culture environment and storage affects the phenotype and clinical utility of MSC-based products, and discuss the techniques better suited to prime MSCs with specific phenotypes of interest and the need for the continued development of standardized assays that provide quality assurance for clinical-grade MSCs. Bioequivalence between cell products and batches must be investigated rather than assumed, so that the diversity of phenotypes between differing MSC products can be accounted for to identify products with the highest therapeutic potential and to preserve their safety in clinical treatments.

In the late 1960s, stromal cells capable of establishing ectopic haematopoietic niches were identified in the bone marrow1 2. Three decades later, the term mesenchymal stem cells (MSCs) was coined3 to reflect the cells’ ability to differentiate into the mesodermal lineage, which includes tissues such as cartilage, bone, muscle, tendon, ligament and fat4 5. Efforts to explore how MSCs could become useful as cell therapies owing to their regenerative and immunomodulatory effects have shown that the mechanisms of action of MSCs are largely mediated by paracrine modes involving microvesicles, exosomes and cytokines6,7. Over 4,000 proteins and 2,400 RNAs are described in the exosome database ExoCarta, opening up possibilities for the discovery of a diverse array of regulatory and cytoprotective actions. Clinical-grade exosomes have been tested in preliminary clinical trials, and, to date, no safety concerns have emerged6–8. Owing to the regenerative potential and potent trophic properties of MSCs and MSC-derived products, they have emerged as a prospective therapeutic tool for numerous clinical applications, and are therefore being extensively evaluated for clinical use9–11

MSCs have been isolated from peripheral blood, adipose tissue, endometrium, amniotic fluid, dental tissues, skin, thymus, spleen, trabecular bone, placental tissue, umbilical cord and Wharton’s jelly9,10,12. Furthermore, cells meeting the minimal criteria for MSCs have been derived from both pericytes and adventitial progenitor cells9,10,12–15. To define these cells originating from diverse tissues, the International Society for Cell and Gene Therapy (ISCT) defined a set of minimal criteria: MSCs grown in classic culture conditions must be plastic-adherent, express high levels of CD105(SH2), CD73(SH3/4) and CD90, and lack expression of CD45, CD34, CD14, CD11b, CD19 and human leukocyte antigen-D related (HLA-DR) surface molecules, and be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro16. In addition to these minimal criteria, additional phenotypic and surface markers have been proposed for the classification of MSC subsets, including stage-specific embryonic antigen (SSEA)-4, platelet-derived growth-factor receptor-α (PDGFR-α), stem cell antigen-1 (Sca-1), nestin, CD44, CD146, CD166 and CD271, among other parameters

such as microRNAs (miRNAs), size, nuclear membrane fluctuations and self-renewal potency9 12 17–19. However, even MSCs meeting the minimal criteria set out by the ISCT16 often display substantial batch-to-batch variation in phenotype and function, differences that are based on donor, tissue source, culture conditions and passage. Variations can also be observed within cell populations20. Unlike pluripotent embryonic and induced pluripotent stem cells (iPSCs), MSCs have limited self-renewal capacity21 and thus fail to meet the classic ‘stem cell’ definition, prompting the use of mesenchymal ‘stromal’ cells as a more fitting name to describe MSCs. Also, unlike iPSCs, MSCs do not form teratomas, a feature that has contributed to their outstanding clinical safety record. Studies of the lifespan of bone-marrow-derived human MSCs isolated from donors of various ages (18–81 years) revealed that, in contrast to mouse MSCs, which readily transform in culture22, human MSCs reach senescence without evidence of transformation23

Clinical safety and progress

Highly publicized concerns about the safety of adult stem cells have primarily been triggered by claims, from private clinics, of the use of ‘stem cell’ therapies entailing only minimal characterization of the cells. The most notable case involved the injection of fractionated lipoaspirate into the eyes of three patients with macular degeneration, which resulted in the loss of vision for these patients24. These careless operations pose serious harm to patients and distract from the progress being made in carefully controlled trials. A meta-analysis of 36 trials, 8 of which were randomized controlled trials, supports the safety of MSC-based treatments25. No association between MSCs and acute infusion-related toxicity, organ-level complications, infection, malignancy or death has been found11,25,26. Over the past few decades, data from hundreds of animal studies have shown that MSCs are highly efficacious in multiple disease models27,28 Several human trials have sought to leverage MSCs to treat diseases of the bone, cartilage, musculoskeletal system, liver and lung, as well as kidney fibrosis, type 1 and type 2 diabetes, Crohn’s disease, cardiomyopathy, myocardial infarction, ischaemic stroke and chronic heart failure9,12,14,29–31. Moreover, the multilineage potential of MSCs,

1Beijing Cancer Hospital, Translational Medicine, Beijing, China. 2Roy J. Carver Department of Biomedical Engineering, Fraternal Order of Eagles Diabetes Research Center, The University of Iowa, Iowa City, IA, USA. *e-mail: jamesyin2010@126.com; james-ankrum@uiowa.edu

their immunomodulation properties and their secretion of antiinflammatory molecules and exosomes make these cells logical candidates for the treatment of immune disorders, including graftversus-host disease (GvHD), inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, Sjögren syndrome, diabetes, amyotrophic lateral sclerosis, systemic sclerosis and systemic lupus erythematosus32–36

Despite intense discussions about the failures of early phase-III studies11 37 38, the large number of clinical trials using MSCs1 39 as well as several recent milestones highlight how the promise of MSC therapy is beginning to be realized. In 2016, Mesoblast licensee JCR Pharmaceuticals received regulatory approval and insurance coverage in Japan to market TEMCELL for patients with steroidrefractory acute GvHD40. In support of this indication, Mesoblast reported that their phase-III acute GvHD trial in the United States, a prospective trial of 60 paediatric patients with steroid refractory GvHD, successfully met its primary endpoint, with 69% of patients responding to Remestemcel-L with either complete or partial resolution of acute GvHD symptoms within 28 days of therapy initiation (ClinicalTrials.gov NCT02336230; ref. 41). Survival data in this cohort are not yet available, but the response rate is highly encouraging and mirrors data previously seen in trials run in academic centres42. Also in 2018, Tigenix’s Alofisel (previously Cx601), an adipose-tissue-derived and expanded MSC product, was approved by the European Medicines Agency for use as a treatment of complex perianal fistula in patients with Crohn’s disease in Europe. In support of the approval, a placebo-controlled trial of 212 patients with Crohn’s disease and treatment-refractory fistulas, local injection of Alofisel resulted in significantly improved remission at both 24-week and 52-week follow-ups43. At the end of the 1-year trial, 56.3% of patients in the treatment group experienced closure of all treated fistulas, whereas only 38.6% of patients in the placebo group did43. Stempeudic’s Stempeucel product also gained regulatory approval from the Drug Controller General of India (DCGI) for the treatment of critical limb ischaemia in patients with Buerger’s disease. In a phase-II trial with 90 patients, patients receiving local injections of the pooled allogeneic MSC product Stempeucel experienced a significant reduction in pain and an improved closure of the ulcers when compared with patients receiving the standard of care44. Although the road towards positive efficacy data in controlled clinical trials and towards subsequent regulatory approval has been rough, the results point to a future when MSC therapies will be available as a treatment for multiple diseases that currently lack effective treatments.

The minimal criteria for MSCs has aided MSC research, yet reliance on these criteria alone has led to the misconception that cells meeting the criteria are equivalent in identity and therapeutic utility. Expectedly, each batch of MSCs exhibits phenotypic features and behaviours arising from their tissue of origin, their subsequent handling in culture and contemporary environmental cues45,46. The approved cell products marketed by Mesoblast, Tigenix and Stempeutics are all sourced and manufactured through distinct processes. Differences in cell source and in culturing practices have real consequences for both tissue engineering and cell therapy, as they influence cell heterogeneity, senescence, secretome and multipotent potential, as well as in vivo homing, survival and integration in damaged tissues. Given that MSCs are influenced by how they are expanded in vitro, researchers in academia and industry working on these cells must recognize that MSCs cultured under disparate conditions generate distinct MSC products, even if they share the MSC minimal criteria. To aid the comparison of different MSC products, bioequivalent standards have been derived from iPSC banks to create a stable and distributable benchmark to which other MSC products can be compared28 47. In this way, potency assays of MSCs derived through new manufacturing processes can be systematically compared to an industry standard for the identification of similarities and differences.

MSCs are more than a collection of surface markers. The following factors should be investigated when expanding MSCs in vitro for application as a cell therapy: (1) the technique used for isolating, purifying or deriving MSCs from different sources; (2) the choice of culture media; (3) the control over the culture device or system; (4) the standardized quality assays for in vitro senescence and genetic stability; and (5) the relevant disease-specific mechanisms of action and potency assays. Most critical among these criteria is the recognition of the need for cell-manufacturing and cell-quality-assessment systems that can ensure maintenance of the MSC properties necessary for the treatment of individual indications rather than a panacea.

integration of MSC culture systems

For MSCs, two crucial issues are the quality and quantity of the cellular product expanded ex vivo. Most clinical protocols to date implement extensive MSC expansion protocols, using a variety of conditions, which change their phenotype and therapeutic potential. To improve MSC research and translation to clinical use, optimized methods to isolate, cultivate, test and use these cells for the treatment of specific conditions are needed (Fig. 1). Integrating these laboratory techniques could enable the standardization of culture systems for the large-scale preparation of MSCs with high quality and purity.

Isolation, purification and derivation. Although cells that meet the MSC minimal criteria can be isolated from diverse tissues, the donor and tissue source influences their functional phenotype; therefore, for certain indications, MSCs isolated from specific tissue sources may be more appropriate. A PubMed search of articles citing MSCs (as of 17 October 2018) shows that the most common source tissue is the bone marrow (~19,000 articles), followed by adipose tissue (~5,800), umbilical cord (~3,600), iPSCs (~1,300) and placenta (~900).

MSCs are positively selected from the bone marrow via their adherence to tissue-culture plastic and their persistence in minimum essential media supplemented with just fetal bovine serum (FBS). Although relatively easy to isolate, the resulting population is heterogeneous and is likely to contain cells that originally resided in anatomically distinct sites of the bone marrow12. Adipose tissue is easier to access in patients, but it is associated with contamination issues similar to those for bone-marrow-derived MSCs. For example, the adipose stromal–vascular cell fraction is composed of preadipocytes, endothelial cells, fibroblasts, immune cells, and several other cell types; any of which may end up in MSC preparations48.

Stromal fibroblasts and dermal fibroblasts adhere to plastic and can express most MSC markers, which is a problem when assessing the purity of MSC preparations49. Also, dermal fibroblasts have some multilineage differentiation potential50, and even the capacity for immunosuppression51,52. Because of the senescence and the tumour-transformation potential of fibroblasts53, contaminating fibroblasts can lead to MSC products with decreased differentiation properties54. Nonetheless, MSCs express significantly higher levels of CD166 and lower levels of CD9 compared with fibroblasts49, a difference that could be indicative of the contamination of MSCs with fibroblasts and could be used to sort MSCs from contaminating fibroblasts by using flow-activated cell sorting (FACS).

In the past decade, a growing number of research groups have turned to more primitive sources of MSCs, such as human umbilical cord and placenta, for a multitude of reasons. Harvesting MSCs from either bone marrow or fat are not insignificant operations for the donor, and the resulting cell product can be highly variable depending on the donor’s age and health55 (MSCs harvested from elderly, diabetic, obese or atherosclerotic patients, for instance, have worse immunosuppressive potential compared with age-matched or young controls56). In addition, the immune-evasion properties

Fig. 1 | the techniques available for the large-scale expansion of MSCs in vitro may lead to diverse changes in cell stemness, heterogeneity and senescence. a, Culture media containing fetal bovine serum (FBS), human AB serum (hAB), human platelet lysate (hPL) or chemically defined media (CDM). b, Culture matrices, such as tissue culture plastic (TCP), extracellular matrices (ECMs), hydrogels and cell-adhesive peptide-bearing thermoresponsive surfaces (such as a poly(N-isopropylacrylamide) matrix (PNIPAm)). c, Distinct culture niches with different dimensions and oxygen concentrations. d, Culture devices applied to MSC expansion, including open and closed culture systems as well as automated and intelligent bioreactors. e, MSC stemness, potency, heterogeneity and senescence are dictated by different combinatorial modes of the four main factors described in a–d. Panels b and the bottom three images in panel d courtesy of ZNB Biological Technology CO., Ltd. Top image in panel d courtesy of PBS Biotech, Inc.

of MSCs enable their allogeneic use, making the requirement for a patient-derived source unnecessary for many applications39

The techniques used to isolate MSCs from human umbilical cord and placenta currently vary widely, with some research groups removing specific sections of the perivascular tissue and others collecting cells from the entire tissue. For umbilical cords, differences in MSCs from different parts of the cord have also been reported (reviewed in ref. 9). Deriving MSCs from the placenta is a relatively recent development, and data have so far been promising. In one of the first human applications of placenta-derived MSCs, the cells showed robust efficacy in treating patients with acute GvHD, with 76% of patients surviving at the 1-year follow-up57. Although the sample size in this study was small, the performance of the cells for treating acute GvHD is among the best reported to date.

More recently, iPSC-derived MSCs have become prominent both as a potential therapeutic product as well as a way to provide a standardized metric to which other MSC products can be compared47 58 . By sourcing MSCs from iPSCs, the issue of donor variability and the risk of depleting a primary stock of cells are eliminated. iPSCs can be propagated and banked, from which new batches of MSCs can then be derived. Once differentiated, iPSC-derived MSCs express the classic MSC markers and proliferate rapidly in culture, but reach senescence after 64 population doublings21. By expanding the quantity of cells before differentiation into MSCs, large numbers of ‘low-passage’ MSCs can be generated for clinical use; this is the model Cynata is employing to generate their Cymerus MSC therapy targeting GvHD and asthma. In addition to therapies, the ability

to distribute large numbers of iPSC-derived MSCs from identical donors and cell batches to laboratories around the world makes iPSC-derived MSCs an appealing candidate to use as a benchmark to compare and contrast alternative MSC isolation, purification and culture techniques47. At this stage, the ISCT minimal criteria are a starting point that has enabled the collection of data on related cells. Still, additional molecular and functional characterizations of different cell preparations are needed to fully understand differences between distinct MSC products, and to help inform the optimal source of cells for each clinical indication.

Culture media. FBS is the most common media supplement in research labs. However, it has several limitations: high cost, batchto-batch variation (arising from different concentrations of its 1,800 proteins and 4,000 metabolites), limited availability (only a third of FBS products meet the regulatory requirements in cell therapy), risk of contamination and transmission of infectious agents, and the potential for triggering xenogeneic immune responses59–61 Therefore, the use of FBS may be deemed inadequate for culturing MSCs intended for clinical application.

To avoid undesirable complications associated with FBS, culture media that can replace animal serum have been developed. The most used are human AB serum (HABS), human platelet lysate (HPL) and chemically defined media (CDM; Fig. 1a)62 63. These alternatives are not without their own limitations, however. Although HABS is routinely tested for viral contamination, it has obvious differences between batches, owing to donor heterogeneity. HPL, prepared by

various release strategies and a suitable alternative to FBS63,64, also has large batch-to-batch variability and an undefined makeup and carries an infection risk similar to other blood-derived products on the market. Moreover, proteomic analysis has shown that HPL obtained from peripheral blood contains pro-inflammatory factors, such as chemokine (C–C motif) ligand 4 (CCL4), metalloproteinase (MMP)-3 and CCL5, that may affect MSC phenotype. HPL may also alter MSC surface marker expression, impairing their immunosuppressive capacity against T-cell proliferation in the presence of an alloantigen and against natural killer (NK)-cell proliferation and cytotoxicity, and can attenuate expression of indoleamine 2,3-dioxygenase 1 (IDO1) when compared with FBS65

A preferred alternative to FBS for the production of clinicalgrade MSCs is serum-free, xenobiotic-free (XF) CDM, which has been shown to improve the multipotent differentiation and expansion rates of MSCs66–68. Of note, several cytokines including the platelet-derived growth factors PDGF-BB, PDGF-AB, transforming growth factor-β1 (TGF-β1), heparin-binding epidermal growth factor (HB-EGF) and insulin growth factor-1 (IGF-1) are known to influence the proliferation, migration and maintenance of multipotent differentiation potential in MSCs69,70. On the basis of these studies, a variety of molecules in addition to cytokines — including basic fibroblast growth factor (bFGF), glucose, vitamins, amino acids, fatty acids and sodium bicarbonate, as well as some chemical agents — have been combined and tailored for the in vitro manipulation of MSCs. Examples of commercialized culture systems using serum-free CDM include RoosterNourish (Rooster Bio), Mesencult-XF (Stemcell Technologies), StemPro MSC SFM XenoFree (Invitrogen), MSCGM-CD (Lonza) and PPRF-msc6, STK1 and STK2 (Abion Inc.)71. In general, these CDM offer multiple advantages for the culture of MSCs for therapeutic applications when compared with conventional serum-based media. Nevertheless, experimental comparisons have suggested that the performance of some human MSC (hMSC) CDM, with respect to the attachment and growth of primary bone-marrow-derived hMSCs, is inferior to FBS-based media. For instance, StemPro MSC SFM supports rapid hMSCs growth at early passages, but the cells then reach senescence earlier (at passage 5) when compared with MSCs grown in control FBS medium72. Similarly, MSC populations cultured in other types of CDM also display diverse changes in hMSC-specific surface antigens (such as STRO-1 CD166, CD73, CD44, CD105 and CD146). Therefore, although progress has been made to move MSC production to chemically defined XF media, the influence of different media on MSC phenotype remains an area of active research, involving continued modification and optimization of MSC media compositions for indication-specific clinical processes.

Hypoxia conditions. Arterial oxygen tension in the bone marrow stays between 10–14%, but the areas within the bone marrow in which MSCs reside are more hypoxic, at 4–7% oxygen tension. Culturing MSCs in hypoxic conditions enhances their proliferative capacity and minimizes their spontaneous differentiation73 Hypoxia (2–5% partial pressure of oxygen ( pO 2)) also enhances cell growth and cell homing, has anti-apoptotic effects by lowering the enzymatic activity of caspase-3, and supports MSC-mediated angiogenesis and engraftment by increasing the expression by MSCs of pro-angiogenic genes such as hypoxia-inducible factor 1α (HIF-1α)/HIF-2α, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1)74 75

HIF plays a protective role as a low-oxygen sensor after ischaemic injury. Upregulation of HIF-1α and HIF-2α enable cells to resist hypoxic stress and suppresses p53 (refs. 74,76). Moreover, it has been observed under hypoxic conditions that population doublings are more frequent as a result of cellular senescence being delayed (cellular senescence, induced by telomere shortening and DNA damage, can be caused under normoxia by reactive oxygen species (ROS),

for example, O2–and H2O2). In addition, hypoxia reduces senescence by upregulating c-jun, dampening the phosphorylation of p38, and increasing the immunosuppressive capabilities of MSCs77. Hypoxic conditioning is thus overall a useful strategy for priming MSCs, for applications in ischaemic and inflammatory environments (Fig. 1c).

Culture matrices. Although multiple choices are available for selecting a surface to support MSC growth, each choice comes with its own consequences, as either soft or stiff culture substrates alter the properties of MSCs (Fig. 1b). MSCs are known to express markers that reflect their substrate. MSCs express neurogenic markers on low-stiffness substrates (0.1–1 kPa), myogenic markers at intermediate-stiffness substrates (8–17 kPa), and osteogenic markers on the stiffest substrates (25–40 kPa)78,79. Other studies have shown that MSCs primed on soft matrices have improved wound healing compared with MSCs primed on stiffer matrices (by reducing granulation tension, myofibroblast content and collagen density, and by increasing angiogenesis); by contrast, stiff-primed MSCs become imprinted by their culture conditions and lead to enhanced scarring in vivo80 81. Analyses of the cytokine production of MSCs have revealed that young MSCs produce less interleukin-6 (IL-6) and more VEGF and glial cell-derived neurotrophic factor (GDNF) when cultured on extracellular matrix (ECM) compared with traditional tissue-culture plastic, suggesting that the phenotype of MSCs is influenced by the culture substrate82. Similarly, the expression of cardiac-specific genes by MSCs can be enhanced by culturing the cells on chitosan membranes rather than on tissue-culture plastic, possibly through the upregulation of Wnt11 expression83

To date, many new culture materials — including hydrogels made with collagens, glycosaminoglycan (0.5–1.5 kPa), methacrylated hyaluronic acid (1.5–7.4 kPa), poly(ethylene glycol dimethacrylate) or alginate84 — can be made with varying degrees of stiffness in order to direct MSCs to differentiate into specific lineages85

In addition to matrix stiffness, the process used to release cells from the growth surface can affect cell viability and differentiation efficiency. For example, poly-ε-caprolactone, a biocompatible and biodegradable material, supports high MSC yields while maintaining their differentiation capability and potency86; after cell expansion, the cells cultured on these ‘microcarriers’ can be implanted in vivo without the need for enzymatic release. Interestingly, some studies have revealed that thermoresponsive polymers (for example poly(N-isopropylacrylamide) and poly(tri(ethylene glycol) monoethyl ether methacrylate) exhibit a reversible temperature-dependent phase transition in aqueous solutions87, a unique property that enables control over cell adhesion and release through external temperature changes88. Moreover, cell-adhesive, peptide-bearing thermoresponsive surfaces have been designed to incorporate a thermally induced ‘on–off’ switch based on integrin–peptide binding. This method enables serum-free cell culture and trypsin-free harvesting, essentially eliminating mammalian-sourced culture components and enzyme-mediated cell damage89, improving the feasibility of fabrication of a cell sheet or of large-scale MSC cultivation. At this time, commercially available thermoresponsive plates and dishes are available on a small scale, but these types of coating are actively being adapted and optimized to meet the needs of the large-scale biomanufacturing processes that are required for cell therapy applications (reviewed in ref. 89).

Culture environments and devices. One of the main risks related to the production of MSCs, as well as of any other cell product intended for clinical use, is microbial contamination; therefore, processes in which sterile product is exposed to air must be performed in a biosafety cabinet meeting the Class 100/ISO 5 air standards, to comply with good manufacturing practice (GMP) guidelines90,91. In addition, the space around the biosafety cabinet should meet Class 10,000/ISO 7 air standards, to minimize the risk of contamination92

In the future, intelligent systems may provide not only GMPcompliant reagents and materials, but also standardized processes with high reproducibility (Fig. 1d). Isolated cells are often cultured on tissue-culture plastic in the presence of different culture media93 94. Plastic culture systems include T-flasks, well plates, Petri dishes, Cellstacks and HYPERFlasks (Corning Incorporated Life Sciences), as well as 10-layer cell factories (Nunc, Thermo Fisher Scientific). However, these 2D culture devices have significant drawbacks90 94: they are labour-intensive, and are susceptible to contamination owing to long-term cultivation and the need for repetitive passaging. More importantly, a low cell-survival rate, the alteration of surface-marker profiles and morphology, and reduced proliferation and differentiation capacity, have been observed after in vitro 2D expansion94,95. Furthermore, this method generates heterogeneous cell populations (multipotent cells along with their progenitor cells and senescent cells) with reduced differentiation potential. All of these factors could result in a decrease in therapeutic success in clinical studies that depend on MSC differentiation as a mechanism of action.

To solve these problems, various 3D in vitro cell-culture systems have been developed to mimic the native microenvironment of cells. By dissecting MSC metabolism, a feeding regimen can be defined and programmed into a process-controlled bioreactor, and optimal culture conditions (such as pH and hypoxic tension) can be selected95–97. Remarkably, the cultivation of MSCs in 3D aggregates has been shown to preserve the cells’ multipotent differentiation potential. Spinner flasks, wavy-walled reactors and rotating-wall vessels, as well as the 3D BioTek Perfusion Bioreactor, are just a few of the bioreactor devices commercially available today90,98 The potential advantages of automated closed-loop bioreactors include: (1) improved expansion and shorter working times, (2) a reduction in operator-related errors and a reduction in batch-tobatch variability, (3) prevention of abrupt fluctuations in pH and in local paracrine environments, which are typically triggered by manual changes of medium, (4) reduced risk of contamination, (5) enhanced traceability due to closed-loop monitoring and control of cell-culture parameters, (6) higher rate of cytokine production, and (7) retained differentiation potential98–100. To fully realize the opportunities provided by automated closed-loop bioreactors, the parameters that lead to a cell product with application-specific potency must be carefully elucidated and validated.

Cryoprotective and post-thawing issues. Allogeneic MSC therapy is dependent on cryopreservation, storage and thawing protocols that maintain the function and potency of the cells. Previous studies have suggested that animal proteins present in serum-supplemented culture media are difficult to remove from cultured MSCs, and that these residual proteins may be immunogenic when injected into patients101. Furthermore, regulatory guidelines demand traceability of all components used in cell manufacturing91, which is often difficult to maintain with pooled animal-derived serum products102 For these reasons, serum-free media is desirable for the cryopreservation of MSCs. Most recently, human albumin and neuropeptides have been used instead of FBS, with MSCs maintaining their cellsurvival and proliferation potentials post-thaw103

An additional concern associated with MSC storage is that the metabolic activity and T-cell suppressive capacity of MSCs can be reduced by cryopreservation104. The immunosuppressive properties of post-thaw cells were previously shown to be impaired in vitro37, leading to upregulated heat-shock proteins, lack of response to interferon-γ-induced upregulation of IDO, and weak suppression of CD3/CD28-driven T-cell proliferation104,105. However, MSCs fully recovered after 1–3 days of post-thaw culture. It is therefore likely that fresh MSCs have superior therapeutic potency when compared with recently thawed cells for some applications106. Additionally, freeze–thaw steps and transient warming also affect MSC potency105;

in this regard, the method for thawing cells post-cryopreservation may have a stronger effect on cryopreservation-mediated cell damage than the media itself107. In a carefully coordinated study, MSCs maintained on dry ice until rapid thaw and plating better maintained viability and expression of the immunomodulatory factors IDO and tumour-necrosis-factor-inducible gene-6 protein (TSG-6) than those exposed to room temperature, which showed progressive decline in all measures107. Also, under tightly controlled freeze–thaw conditions, MSCs display reduced viability and T-cell suppression ability in vitro, yet maintain in vivo potency when applied to a mouse model of ischaemic stroke108. Furthermore, IFN-γ priming (which had been proposed to protect against cryo-induced damage109) diminished the therapeutic efficacy of MSCs in treating ischaemic stroke110. Therefore, it is necessary to develop comprehensive and optimized cryopreservation, storage and thaw protocols, and to identify assays that predict MSC potency for the treatment of specific conditions111. Interestingly, therapies developed and sold by JCP Pharmaceuticals, Mesoblast, Tigenix and Stempeutics are all cryopreserved MSC-based therapies. Taken together, distributing best practices for MSC preparation and creating relevant facilities for moving cells in and out of cryopreservation are essential endeavours for the successful clinical translation of MSCs.

As cell therapies have progressed, encompassing chimeric antigen receptor T-cell (CAR-T) therapy and in vitro fertilization and progenitor cell therapies, so has research into optimization methods for the preservation of living cells, such as vitrification and lyophilization. Although cryoprotectant-mediated, controlled-rate slow freezing is widely used, technological advances (such as the Prestige Lyotechnology platform, developed by Osiris Therapeutics, for the lyophilization of viable cells112) may provide superior alternatives. Whether these technologies can produce cells at a scale, quality and efficiency that is superior to traditional systems is unclear, but the rise of cell therapies is sure to catalyse these and similar efforts.

Quality criteria for the evaluation of MSCs

As MSC products are scaled up in large manufacturing workflows to generate an appropriate cell product for clinical applications, there is a need to provide quality assurance for each batch of cells. Functional potency assays, in vitro or in vivo, are ideal to demonstrate differentiation45 or immunomodulatory potential113,114, but are time- and resource-intensive. To address this need, researchers have focused on the identification of surrogate markers of MSC quality associated with MSC function that can rapidly be assessed on each batch of cells.

Cell size. MSCs expanded in FBS-supplemented media undergo a transition from predominately spindle-shaped at early passages to larger fried-egg-shaped the longer they are maintained in culture71 This increase in size can have consequences on the subsequent application of the therapy.

The most common route of MSC delivery is intravenous infusion, however, it can result in MSCs being lodged in the microvasculature of numerous organs115. Entrapment of MSCs within the capillaries of the lung, in particular, may cause serious adverse events. For this reason, small size is desirable when MSC products are intended to be administered systemically. Moreover, the small size of MSCs correlates with their differentiation potential, which is often a crucial factor for cell-based therapy100,116,117. A recent study indicated that 3D culture reduces MSC size by promoting vesicle secretion116. A similar effect can be achieved by treating MSCs with cytochalasin D or by blocking integrin β1. In addition, a few original culture methods improve the expansion of MSCs and even rejuvenate senescent MSCs100,116 With multiple observations indicating that small size correlates with improved MSC function, this characteristic should be considered as part of the quality criteria of the product100,117. In this regard, a report by the US Food and Drug Administration has shown that measuring

Table 1 | Potential functions of MSC surface markers

Marker Function references

CD29 Exosome uptake, cell migration 125,154

CD44 Homing and adhesion 119,120

CD73 Anti-inflammatory 124–126

CD90 Stemness 120

CD105 Antagonization of the binding of TGF-β 1 to its receptor 122

CD146 Anti-ageing, stemness 126,127

CD166 Commitment and differentiation 119

HLA-A,B,C Resistance to NK-cell-mediated lysis 127

SSEA-4 Stemness 100,122,138

21 morphological features extracted using an automated imageprocessing software enabled researchers to predict which batches of MSCs would retain high immunosuppressive potency in co-cultures with T-cells118, laying a path for the multiparametric morphological profiling of MSCs to be used as batch-specific quality metrics. Validation of this approach in the context of predicting MSC potency in a specific disease model or clinical trial would further support the use of these in vitro predictive assays for MSC therapy.

Surface markers. The expression of specific combinations of surface molecules in MSCs may be used as an indicator of the cells’ differentiation potential, lineage commitment, senescence and therapeutic function (Table 1). The high expression of specific cluster of differentiation (CD) molecules on the surface of MSCs may be associated with the maintenance of multipotent differentiation potential (for example, the expression of STRO-1, SSEA-4, CD44, CD73, CD90, CD105, CD146, and CD169; refs. 12,17,119).

It has been suggested that CD90 maintains the multipotency of MSCs by impeding differentiation commitment120; CD90 correlates with the undifferentiated status of MSCs, and a decrease in its expression has been seen to be associated with enhanced osteogenic and adipogenic differentiation of MSCs in vitro.

CD44, a hyaluronate receptor also known as homing cell-adhesion molecule (HCAM), is widely expressed on several types of stem cell119,121. It has been suggested that changes in CD44 and CD166 expression in MSCs could indicate that the cells are more prone to differentiation119. In addition, the level of CD105 expression in MSCs cultured in HPL media is lower than in those cultured in DMEM/FBS65,122

Another marker of interest, the surface ectonucleotidase CD73, has been associated with enhanced adenosine production and increased T helper (TH)1 cell apoptosis, mediated by CD73containing MSC-derived exosomes that suppressed GvHD123,124 CD73 is a key factor in the metabolism of pro-inflammatory adenosine triphosphate (ATP) into anti-inflammatory adenosine125.

Furthermore, the expression of melanoma cell-adhesion molecule (MCAM, also known as CD146) has been shown to gradually decrease after multiple passages of MSCs from human umbilical cord blood126. A gradual decrease in CD146 expression would therefore be an indication of the ageing of these cells. Furthermore, increased expression of human leukocyte antigen (HLA)-ABC on MSCs confers resistance to NK-cell-mediated lysis127, and could thus be used as a surface marker of in vivo persistence. Taken together, by moving beyond the binary assessment of the minimal criteria markers, the identification of nuanced differences in the phenotype of MSCs harvested from different tissues, grown in different media, or primed with different strategies, may predict the behaviour and suitability of MSCs for specific clinical applications.

Gene-expression profiles and genetic stability. Following culture in FBS or HPL, MSCs undergo distinct changes in gene-expression patterns128. In fact, all MSC preparations undergo significant geneexpression shifts on long-term culture, with genes involved in differentiation, senescence and cell death upregulated, and genes involved in proliferation downregulated. Gene-expression profiles have revealed that the in vitro expansion of MSCs derived from Wharton’s jelly for 12 passages promotes the selective overexpression of 157 genes and the downregulation of 440 genes129 (when compared with the fourth passage). Thus, MSCs seem to age during in vitro expansion. Similarly, proteomic analysis has shown the presence of an ageing phenotype in MSCs during in vitro expansion130. Furthermore, microarray analysis has indicated that some miRNAs are also significantly different between early and late passages. These changes in gene-expression patterns are in agreement with the functional differences observed following repeated passaging; for instance, MSCs from late passages express lower levels of immunomodulatory IDO131 and are less able to control GvHD symptoms in clinical trials132. In addition, high-passage MSCs lose their multipotent differentiation potential133 and display reduced growth kinetics134. Therefore, the monitoring of relevant RNA, miRNA and proteins, in addition to the minimal criteria, may assist in the evaluation of changes in MSC phenotype when implementing variations in culturing procedures. The plasticity of the MSC phenotype in culture necessitates a careful analysis of the changes in MSC identity that occur as a result of modifications in culture processes, as these may have detrimental impact on the clinical application of the cells.

The spontaneous differentiation of MSCs during extended culture expansion poses a potential hazard for cell transplantation. FACS analysis has shown that MSCs at passage three are more homogeneous compared with those at passage nine, and that the intracellular complexity of MSCs keeps varying with consecutive passages135. Moreover, MSCs at passage nine show a significant increase in the frequency of formation of nucleoplasmic bridges, indicating the occurrence of genetic rearrangements, including dicentric and ring chromosomes136. Therefore, to ensure the safe clinical application of MSCs, the cytokinesis-block micronucleus assay should be included in the release criteria for batches of MSCs produced for clinical use.

Ageing and senescence. Recent studies have shown that MSC quality declines with age137, as continuous subculture leads to changes in cell morphology, cell enlargement and ultimately senescence138–140 Since MSCs need to be expanded in culture to obtain therapeutic doses, cellular senescence is inevitable under traditional culture conditions. In senescent cells, changes in gene expression mediated by the IL-1α–miR-146a/b–IL-6–C/EBP-β pathway and the related AKT–p38–NF-ĸB- and mTOR-mediated pathways result in the senescence-associated secretory phenotype (SASP) and in increased activity of β-galactosidase141,142. SASP contains tumour necrosis factor (TNF)-α, IL-6/8, matrix metalloproteinase-3 (MMP3), monocyte chemoattractant protein-1/2 (MCP-1/2) and IGF binding proteins (IGFBPs142,143). Therefore, the infusion of senescent cells into patients may attract immune cells, or induce malignant phenotypes in neighbouring cells144. Low glycolytic activity and diminished T-cell suppression has been observed in senescent MSCs145 Furthermore, tunable resistive-pulse-sensing analysis has demonstrated that microvesicles (MVs) derived from senescent MSCs are clearly smaller than those from younger MSCs, whereas a decrease in CD105 and bulk miRNAs with an increase in miR-146a-5p has been reported on MSC-MVs during MSC senescence146. Functional analysis has revealed that miR-146a-5p downregulates most of its target genes found in MSC-MVs and MSCs during senescence147. Nevertheless, several experiments have revealed that cultures using STK2 medium or decellularized bone marrow-derived extracellular

MSCM

Rejuvenation

Criteria for high-quality MSCs

Homogenous MSCs

Small size (15–20 μm)

Spindle type

Spiral growth

Positive CDs >98%

Negative CDs <1%

Low β-galactosidase

Low ROS

Low SASP

Low p16, p21, p53

Low NPBs

Low DNA damage

High growth rate

High differentiation potentials

High CFU-F efficiency

Strong immunomodulation

Strong paracrine ability

Fig. 2 | Morphology of MSCs in culture. Observed at 5× magnification on an inverted phase-contrast microscope. Top: human adipose-derived MSCs (AD-MSCs) were subcultured in 10% FBS medium from passage 2 (P2) to passage 12 (P12). Middle: AD-MSCs were expanded in a rejuvenating medium (MSCM) from P2 to P12. Bottom: senescent AD-MSCs (left) were rejuvenated by MSCM (right). The recommended criteria for highquality MSCs used for cell therapy are also given. Positive clusters of differentiation (CDs) include CD166, CD146, CD105, CD90, CD73, CD44, HLA-A,B,C and CD29; negative CDs include CD31, CD34, CD45, CD14 and HLA-DR. Parameter levels in the late passages can be obtained by comparing them with those in the first two or three passages. ROS, reactive oxygen species; SASP, senescence-associated secretory phenotype; NPB, neuropeptide B; CFU-F, colony forming units–fibroblast.

matrix (BM-ECM) from young donors reduces and may even reverse the cellular senescence of MSCs71,148, and that MSCs obtained from elderly mice and then cultured on BM-ECM produced by young mouse stromal cells restored replicative and osteogenic potential89. In this context, a ‘rejuvenating’ medium (named MSCM) containing highly purified HPL and several defined chemical components has been developed (www.genobio.cn; Fig. 2). Because MSC senescence caused by in vitro expansion or human ageing is a critical issue that needs to be addressed in order for MSCs to be used for cell-based therapy, the implementation of methods for evaluating MSC senescence during MSC quality control would be informative (Table 2).

Exosomes. In addition to growth factors, morphogens, chemokines, cytokines, microvesicles and glycosaminoglycans, MSCs also produce exosomes. MSC-derived exosomes enable the transfer of proteins, mRNAs and regulatory miRNAs to host cells to exert a multitude of biological effects6 8 149 150. Similarly to the other soluble factors produced by MSCs, MSC exosome production is highly dependent on the cells’ origin and contemporary signals. For example, MSC-derived exosomes can differ in content, potency and release quantity depending on the source tissue151. Furthermore, it has been shown that exosomes produced by MSCs from bone marrow with multiple myeloma altered miRNA content compared with MSCs from healthy bone marrow152. A multitude of studies have demonstrated MSC-derived exosomes have much of the same influence on immune effector cells as live MSCs149,153. Interestingly, radiation therapy, which is common before bone marrow transplantation, increases the colocalization of CD29 and CD81, resulting in enhanced uptake of MSC-derived exosomes154. The content of exosomes can also be influenced via priming with cytokines: for instance, TNF-α-exposed MSCs produce exosomes with enhanced bone regenerative potency compared with unprimed MSCs120. MSC-derived exosomes have shown promise for regenerative medicine, but owing to the heterogeneity of the MSCs themselves

Table 2 | Characterization and phenotypic properties of senescent MSCs

Senescence indicators references

Enlarged cells with a fried-egg-like morphology 71,116

Low expression level of CD146, CD105 and SSEA-4 100,122,127,138

Higher β-galactosidase 143,145

Higher concentrations of intracellular reactive oxygen species

Stronger senescence-associated secretory phenotype (SASP) 142,146

Higher concentrations of miR-146a-5p in MSC-MVs

Increased formation frequency of nucleoplasmic bridges 136

Low expression of stemness markers 145

Higher expression of the senescence markers p16, p21 and p53 140

Enlarged γH2AX foci and 53BP1 foci

Decrease in growth rate, multilineage differentiation, immunomodulation and migration and homing abilities 144,148

Increased tumour-promoting functions 148

standardized characterization and potency assays are also needed for MSC-derived exosome products.

Priming MSCs for clinical applications

Similarly to immune cells155, MSCs have been shown to ‘remember’ a stimulus after transitioning to new environments122,156. Therefore, MSCs have been primed to trigger a ‘short-term-memory’ effect (mimicking microenvironmental stimuli) in vitro, thus avoiding the need for in vivo activation of the MSCs when aiming towards specific therapeutic activities.

Inflammation and immune modulation. The anti-inflammatory properties of MSCs can be enhanced to promote the efficacy of tissue repair. Although inappropriate culture conditions can cause a reduction in the anti-inflammatory ability of MSCs, MSC multicellular spheroids produced in 3D scaffolds and media have higher therapeutic potential through the upregulation of TSG-6 and stanniocalcin-1 (STC-1) expression after transplantation157,158 Moreover, activated MSCs secrete prostaglandin E2 (PGE2), which suppresses pro-inflammatory M1 macrophages, tilting the balance between M1 and anti-inflammatory M2 macrophages159,160 (Fig. 3). Simultaneously, activated MSCs secrete TSG-6, which dampens Toll-like receptor (TLR)-2/NF-ĸB signalling in macrophages to decrease production of pro-inflammatory mediators161. Although advantageous for the suppression of macrophages, the potency of the spheroid phenotype to modulate other types of immune cell is less understood.

Similarly to what occurs in immune cells, in MSCs the reduction of NF-κB expression suppresses the pro-inflammatory cytokines IL-6, IL-8, TNF-α and MCP-1 (refs. 131 162 163). Interestingly, anti-inflammatory factors secreted by MSCs (including IL-4, IL-10, GDF-15, ICAM-1, IL-1 receptor antagonist, TNFR1, CCL-20, TFF-3 and thrombospondin-1) are significantly increased in specific culture conditions71. It may be feasible to convert MSCs into anti-inflammatory memory cells in vitro through the selective activation of anti-inflammatory genes or via the selective inhibition of inflammatory signals for reducing NF-κB activity, which can be achieved through gene therapy131,163,164.

In vitro experiments have revealed that MSCs inhibit T-cell and B-cell activation and dendritic-cell differentiation, impair the

TSG-6, STC-1, PGE2, IL-1ra, CCL-20, IDO

IDO, IL-6, iNOS, HGF, HLA-G, PD-L1, PGE2

CBP/p300 Ac Ac

Angiogenin, cystatin C, G-CSF, GM-CSF, M-CSF, HGF, MIF, PDGF-AA Signalling molecules

IGF-1, bFGF, VEGF, HIF-1α, HO-1, STC-1

Anti-inflammation

Immunomodulation

Anti-apoptosis

Fig. 3 | Potential mechanisms of MSC-primed therapeutics. MSCs can be primed via different signals (such as hypoxia, matrix mechanics, 3D environment, non-coding (nc) RNA, cytokines and hormones) to acquire and retain phenotypes relevant for therapeutic applications. The potential mediators of the short-term memory of those phenotypes include a vast array of stimulus-responsive regulatory molecules within MSCs, such as surface receptors and antigens, inducible regulatory molecules such as miRNAs, signalling molecules and transcription factors, and a subset of newly deposited chromatin modifications, such as DNA methylation and histone acetylation. These primed MSCs can remain sensitive to a specific stimulus for a short time, thus eliminating the lag time for activation in vivo after engraftment. Primed MSCs promote tissue repair through cell differentiation and replacement, and also via the delivery of bioactive factors through different secretory modes. dsRNA, double-stranded RNA; VPA, Valprioc acid; CBP/p300, CREB-binding protein/E1A-binding protein p300; Ac, Acetyl group; iNOS, inducible nitric oxide synthase.

cytolytic potential of NK cells, and lead to an increase in the numbers of regulatory T cells165 (Treg). In the clinic, acute GvHD patients treated with early-passage MSCs survived longer than those treated with late-passage MSCs132. However, in experimental autoimmune encephalomyelitis, a preclinical model of multiple sclerosis, the potency of MSCs seems to be linked to the tissue and donor source of the cells166, suggesting that active signalling through trophic factors plays a role in MSC phenotype. Consequently, the ability to control the composition of trophic signals originating from MSCs has been widely explored. For example, valproic acid enhances the immunosuppressive potency and metabolic activity of human MSCs167. It has been proposed that MSCs can be induced into MSC1 and MSC2 phenotypes through the activation of TLR-3 or TLR-4. For example, the TLR-3-primed MSC2s can suppress T-cell proliferation and induce Treg cell generation by producing immunomodulatory factors such as IDO and TGF-β1 (refs. 165 168).

IFN-γ activates the transcription and synthesis of IDO-1, hepatocyte growth factor (HGF) and TGF-β in MSCs through JAK/ STAT signalling169 170. It has recently been shown that MSCs treated with TNF-α and IFN-γ for 24 hours were resistant to the lipotoxic effect of palmitate, and that the effect of the brief priming was still observed 4 days after removal from the priming medium170

The primed cells maintained the ability to block T-cell proliferation and cytokine production even in the presence of a challenging metabolic environment. Likewise, sustained IDO expression has been achieved by presenting IFN-γ to MSCs via microparticles that resulted in a sustained improvement in the MSCs T-cell suppressive phenotype171

Additionally, both IL-10 and progesterone can enhance the immunosuppressive effects of MSCs through the activation of HLA-G (ref. 172). Intriguingly, the transient stimulation of adiposederived MSCs with TNF-α or lipopolysaccharide was shown to dramatically increase the secretion of MCP-1, IL-8 and IL-6155, and that the cytokines are quickly released following a second round of stimulation. In these experiments, miR-146a, miR-150 and miR155 were suggested to be crucial factors in immunomemory. Of note, 512 immunomodulation-related genes have been found to be upregulated in IFN-γ-preconditioned MSCs173.

To take advantage of the cytokine-mediated conditioning of MSCs, the in vitro pre-activation of the cells harvested at a high density may be a practical strategy to maximize the therapeutic effects of MSCs with improved immunosuppressive properties169,174. Interestingly, epithelial stem cells can be primed with an initial stimulus to open up key chromosomal stress-response elements that

Adipocyte Osteoblast Myocyte Neuron Chondrocyte

then respond rapidly on secondary stimulation. Similar mechanisms regulating the important IDO promoter region have been recently discovered in MSCs163 and other conditioned responses may exist for other MSC mechanisms of action. Careful mechanistic studies of how MSCs regulate and maintain their therapeutic phenotype is needed in order to generate priming strategies specifically tailored to control MSC potency for specific disease indications.

Countering apoptosis. The poor survival of transplanted cells is a limitation for clinical indications that depend on long-term engraftment. MSC apoptosis is accompanied by decreased functional activity under oxidative stress and inflammatory conditions. Hypoxic preconditioning of MSCs within 3D culture systems has been shown to improve the therapeutic efficacy of MSCs on transplantation175. MSCs derived from adipose tissue and cultured in 3D spheroids express high levels of HIF-1α and manganese superoxide dismutase and are resistant to oxidative stress-induced apoptosis176. Pre-treatment with 17β-estradiol protects MSCs from H2O2 exposure in vitro, which may improve cell survival following recruitment to injured tissues in vivo177. Priming MSCs with oxytocin seems to increase MSC survival in hypoxic environments through the activation of the enzymes AKT and ERK1/2178, whereas exposure of hMSCs to apoptotic cytokines stimulates hMSCs to secrete STC-1, a peptide with anti-apoptotic effects178.

MSC susceptibility to NK-cell-mediated lysis can be reduced by IFN-γ stimulation179. Similarly, preconditioning MSCs with pro-survival factors, or overexpressing protective molecules, such as SERPINB9, can be used to enhance MSC resistance to NK-cellmediated killing180. Moreover, culturing MSCs in 3D collagen scaffolds or on plates coated with laminin or hyaluronan results in significantly enhanced secretion of anti-apoptotic factors181. Of interest, miR-21, an anti-apoptotic miRNA, is abundant in MSC exosomes182. MSCs can also engulf foreign mitochondria, which leads to the induction of heme oxygenase-1 (HO-1) and mitochondrial biogenesis, which aid in cell survival183. Thus, a better understanding of how the cytokines present within inflammatory environments in vivo modulate MSCs could be helpful to develop more effective priming strategies for MSCs in culture.

Repair ability. The MSC secretome contains factors that can suppress local immune responses and reduce oxidative stress, fibrosis and cell death184. The secretome, however, is also influenced by the MSC source and culture conditions used to expand the cells. Factors related to cell repair, including angiogenin, cystatin C, colony-stimulating factors (CSFs, such as G-CSF, GM-CSF and M-CSF), hepatocyte growth factor (HGF), macrophage migration inhibitory factor, PDGF-AA and lipocalin-2 are increased in culture when using STK2 media compared with FBS-based media71 GRO-α, HGF and IL-8 produced by MSCs conditioned by hypoxia has been reported to reduce cardiomyocyte apoptosis and ROS production, improving their metabolism and function185. MSCderived exosomes have also been shown to transfer IGF-1R mRNA to cisplatin-damaged proximal tubular epithelial cells, making the cells responsive to the protective effects of native IGF-1 (ref. 186). Other studies have reported that miR-494-containing MSC exosomes protect against ischaemia–reperfusion-induced cardiac injury, suggesting that miR-494 in MSC-derived exosomes could promote tissue repair187,188

Homing. Depending on the disease and mechanism of action, MSC homing to a site of injury may be required to elicit a therapeutic effect115,189. MSCs have an innate ability to home to sites of inflammation190–192 and to transmigrate through inflamed endothelium193 194 via a process that is analogous, although less efficient, to that for leukocytes195. Owing to the low efficiency of this process, MSCs have been engineered or primed for enhanced homing capacity. The

homing of cells to target tissues is achieved via the expression of different combinations of chemokine receptors (CCR2, CCR4, CCR7, CCR10, CXCR4, CXCR5, CXCR6, CXCR7, CD29, CD44 and ULBP1), as well as via MMP-1/2/9, by the incoming cells189,194,196–198 (Fig. 3). Factors that promote cell migration, including stromal cellderived factor 1 (SDF-1), HGF, IL-6, EMMPRIN, G-CSF, IGF-1, IFN-γ, PDGF, vascular cell adhesion molecule 1 (VCAM-1) and E-selectin, are often increased in damaged tissues119,189,194,196–198 Freshly isolated MSCs have greater homing efficiency than cultured cells, as cultured MSCs gradually lose expression of factors supporting migration into tissues191. Similarly, extensive passaging decreases the engraftment efficiency of MSCs194

Higher expression of CXCR4, MMP-2 and MMP-9 can be obtained for hypoxia-derived or spheroid-derived adipose tissue MSCs, compared with the use of conventionally cultured cells197 Hypoxia can thus also be used to promote MSC homing: for instance, when MSCs cultured under hypoxia are transfused into diabetic mice, they migrate to damaged sites, actively modulate the local inflammatory environment and contribute to tissue repair196–198 Interestingly, erythropoietin can provide a strong stimulus to bonemarrow-derived MSCs for kidney infiltration199, whereas MSCs engineered with modified surfaces show greatly enhanced migration to sites of inflammation200–202. In a lipopolysaccharide-induced inflamed ear murine model, hyaluronic-acid-treated MSCs demonstrated significantly higher inflammatory targeting mediated by the overexpression of CD44 (ref. 121). Furthermore, MSCs with a high expression of CD49d/CD29 and CD44 can cross the endothelial barrier more effectively when VCAM-1 and E-selectin expressed in endothelial cells become activated under injury and inflammatory conditions203 204. Finally, transplanted MSCs may enhance the recruitment of host MSCs to sites of injury through the production of mobilizing factors such as HGF, leukaemia inhibitory factor, SDF1, stem cell factor and VE-cadherin (refs. 189,194). For MSC-based therapies that rely on migration of cells towards a specific site, MSC homing potency needs to be evaluated before clinical application189.

MSC differentiation and commitment. The stiffness of the culture matrices used for MSC expansion has a strong effect in determining the differentiation pathways of the cells. Soft matrices (elastic modulus, 0.1–1 kPa), stiff matrices (8–17 kPa) and hard matrices (25–40 kPa) induce transcriptional markers of neurons, muscles and osteoblasts, respectively78. By altering chromatin structure, lineage priming makes specific regions of the DNA open and available for key transcription factors and miRNAs to bind while closing other segments that may be required for differentiation into other lineages205. For example, cyclic tensile loading, a mechanical method utilized to promote neuronal transdifferentiation of MSCs, works partly through the regulation of Rho GTPase activity, a target of miR-124 (ref. 206; the expression of miR-124 is progressively increased during the development of the central nervous system207). In addition, the overexpression of miR-21 in MSCs primed in soft matrices facilitates osteogenic differentiation, whereas the downregulation of miR-21 in MSCs primed in stiff matrices enhances MSC adipogenesis80. Putting this observation into practice, priming MSCs on soft substrates or erasing ‘mechanical memory’ through downregulation of miR-21 can prevent MSCs from becoming pro-fibrotic in vivo80. Additionally, stiff substrates have been shown to lead to an increase in MSC expression of miR-100-5p and miR-143-3p, a decrease in mTORC1 activity and a bias towards osteogenic differentiation, whereas soft substrates generate the opposite signature and are biased towards adipogenic differentiation208. Therefore, miRNA signalling in hMSCs may be employed to modulate hMSC differentiation fate for tissue regeneration and cell therapy.

In vitro priming and conditioning can be used to coach MSCs towards shifting their phenotype in therapeutically desirable directions. Future work in the development of priming strategies should

aim to determine the durability of the priming strategy — that is, once the cells are transferred to a new environment, as happens during transplantation, it is critical to assess whether the cells retain the primed phenotype and when they eventually adapt to their new environment. The timescale of MSC memory and adaptation is important, because for many applications of MSCs, cells are shortlived, lasting only a few days, and thus even short-term memory may be highly advantageous for the improvement of control over cell therapies. For example, it has been shown that MSC priming with IFN-γ to upregulate IDO expression augments immunosuppressive potency131, even after cryopreservation110. We suggest that, to tailor the efficiency of MSC therapy for specific applications, the abilities of MSCs should be evaluated by predictive in vitro functional assays before the cells are used in clinical applications. Importantly, multivariate potency matrices are likely to be required in order to robustly predict in vivo function113 118 209 210. The ability of MSCs to retain functions triggered by environmental signals may be used to modulate them better to suit specific clinical applications: primed cells may be able to migrate and adapt to the microenvironments of inflamed or injured tissues more rapidly, exhibit more powerful therapeutic effects, and replace and repair damaged tissues and organs with greater efficiency.

outlook

The development of standard and normalized therapies using MSCs and MSC-derived progenitors, including the identification and evaluation of the most reliable MSC isolation methods, optimized cell culture systems, and methods to derive sufficient numbers of high-quality MSCs, are crucial steps towards enabling investigators to effectively compare data and to develop superior MSC-based therapies. Despite the steady progress made in basic research and in the clinical application of MSCs to date, several important questions remain with respect to the preferential use of homogeneous versus heterogeneous MSCs, the advantages of allogeneic versus autologous cells, and the cell-replacement and paracrine roles of MSCs. Efforts to define the phenotypic characteristics of heterogeneous MSCs were initially stymied by their puzzling properties19 As it became clear that purifying a population of MSCs using only one marker is impossible, MicroSAGE analysis demonstrated that a clonal colony of human MSCs express mRNAs characteristic of endothelial, epithelial, mesodermal and other lineages211. Similarly, FACS analyses have shown that MSCs exhibit a subset of markers derived from multiple cell lineages12,17,18,212. All of these observations underscore the fact that MSCs can give rise to many types of functional cell, and therefore that they have a stem-cell-like signature. However, MSCs are not a homogenous population of cells, given that the cells are easily polarized into distinct phenotypes by TLR or cytokine exposure, and that they manifest different degrees of heterogeneity when placed in different environmental niches164. MSCs also have some degree of plasticity, as studies have found that optimized culture systems can reverse heterogeneous MSCs into a population of relatively homogeneous cells with similar size, phenotype and differentiation potential71,143.

Whether to use autologous or allogeneic MSCs for therapeutic applications remains a point of debate. Human autologous and allogeneic MSCs typically require in vitro expansion to generate clinically relevant cell numbers. Allogeneic MSCs may thus be more attractive, as the ability to expand and bank cells in advance enables their immediate off-the-shelf use. However, although the prevailing dogma has been that allogeneic MSCs are immune-privileged213, mismatched MSCs are only immune-evasive39 and can be immunogenic. For example, while culture-expanded MSCs express low levels of HLA-A,B,C and are negative for HLA-DR, MSCs stimulated with IFN-γ or differentiated into mature cell types express high levels of both214, suggesting that allogeneic MSCs can evoke an immune response that leads to cell rejection, as has been observed

in clinical trials39,215. Moreover, allogeneic MSCs can be rapidly phagocytized by monocytes residing in the lungs and other body sites216. By contrast, autologous stem-cell-based therapies are better positioned to engraft and to provide long-term support to damaged tissues, given their low immunogenicity. Autologous cells may give rise to different types of functional cells, replacing those damaged or lost in injury sites217. To enable MSCs engrafted at injury sites to differentiate into functional cells, thus promoting tissue restitutio ad integrum, it may be necessary to make MSCs committed to specific types of progenitor before delivery. This is practically complicated as it requires the generation of ‘personalized MSC products’, which is time consuming if culture expansion is required, and can lead to significant variability in the resultant cell product arising due to age or co-morbid conditions.

A related question is whether the observed beneficial effects of cell transplantation are due to mechanisms that rely on integration and differentiation of transplanted cells, or to purely trophic mechanisms mediated through cytokines, exosomes and the broader secretome of the transplanted cells. The current view of MSC functionality holds that MSCs heal injured and diseased tissues via comprehensive modes rather than solely by long-term engraftment and differentiation8,149. These modes of repair include cell fusion, trophic effects of growth factors, cytokines and hormones, cell–cell interactions mediated by tunnelling nanotubes, the release of exosomes and microvesicles, and the activation of autophagy6–8,150,217 A critical next step for the field of MSC therapy is to identify the dominant mechanisms of action that MSCs employ to facilitate healing in individual diseases. The rise of MSC-derived exosomes has shown how much of the therapeutic potency of MSCs can be achieved without the need to deliver living cells, potentially simplifying the logistics and reducing the costs associated with living therapeutics. The precise conditions that can be treated with this strategy, however, are not yet fully known.

Multilineage differentiation potential is required to meet the MSC minimal criteria, yet it is a liability for many clinical applications. The use of MSC differentiation commitment, particularly its bias of directional differentiation and potential to give rise to specific cell types of interest, is one of the most promising areas of research in tissue regeneration and disease treatment218,219. There is a growing interest in the use of MSCs (or engineered MSCs) and their descendant progenitors in the development of tissues, in degenerative diseases and in therapeutic applications220. However, if MSCs are being developed to treat neurodegenerative disorders, such as Parkinson’s disease, then their osteogenic and adipogenic potentials are only a hazard. Thus, in the derivation of MSC products for specific applications, it is sometimes appropriate to prime MSCs to lose certain MSC-defining characteristics. Rather than focusing on minimal criteria, as MSC products get closer to clinical application the focus must turn to release criteria that are known to correlate with the safety and efficacy of the product, for each disease.

A final point of crucial importance concerns the in vivo administration modes of MSCs. It remains unclear how the frequency, timing, route and dose of administration affect MSC-based therapies. For disease indications requiring tissue targeting, it is critical to bypass the initial lung entrapment by decreasing MSC size and enhancing organ-specific capture through the modulation of cell-surface properties115,221. And pharmacokinetic models of the in vivo dynamics of systemically delivered MSCs suggest that they be administered more frequently and at larger doses, to maximize their therapeutic benefit222. Although recent clinical successes have brightened the future of MSC-based therapies, a large number of variables with a significant impact on the success of clinically translated MSC products remain to be explored. The potential of MSC therapies will be realized only by developing high standards for the handling of MSCs, from isolation to infusion.

Review ARticle

Received: 19 March 2018; Accepted: 14 November 2018; Published online: 28 January 2019

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Acknowledgements

This work was supported by grants from the National Key Scientific Program (2017CB964902) to J.Q.Y. and J.Z.

Author contributions

J.Q.Y., J.Z. and J.A. conceived of and wrote the manuscript. J.Q.Y. and J.Z. generated the figures.

Competing interests

J.Q.Y. was involved in the production of the rejuvenating MSCM media, marketed by Geno Biotechnology Co. in China. J.A. and J.Z. declare no competing interests.

Additional information

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