The therapeutic potency of ASCs presupposes the presence of as many functional cells as possible in the damaged tissue. To achieve this requirement, many scientists sought to optimize the manipulation and administration methodology of naïve ASCs, including source differences, delivery timing, administration route, and dosage. Nonetheless, these attempts did not yield the desired improvement. Therefore, there is a need for novel strategies to enhance the capability of survival, homing to the site of inflammation, and immunomodulatory properties. Until now, four different approaches with encouraging outcomes were proposed in ASC therapy: pre-conditioning with various bioactive molecules, genetic engineering of functional genes, modification of culture condition, and direct application of extracellular vesicles. In this section, we discuss and update each ASC enhancement strategy according to the categories.
3.2. Genetic Manipulation
Gene therapy is generally defined as the experimental technique that introduces exogenous DNA into a patient’s cells or directly in vivo to treat a variety of inherited and acquired genetic disorders. In recent years, the ease of MSC genetic manipulation was reported in vitro, and MSCs may serve as a suitable delivery vehicle for gene therapy [108
]. Indeed, genetically modified MSCs, particularly overexpressing suicide genes, were used in glioblastoma repression [110
], and genetic engineering can also be applied to transduce functionally critical genes directly into the ASCs themselves to enhance the therapeutic potency. To date, the genes responsible for survival, migration, and immunomodulatory properties were mainly targeted in ASC therapy and, overall, genetically modified ASCs were found to be more effective than wild-type cells [111
]. Here, we introduce several reports on enhancing the function and therapeutic effect of ASCs through a genetic engineering approach.
Pre-conditioning strategies to strengthen adipose-derived stem cell (ASC) function.
Pre-conditioning strategies to strengthen adipose-derived stem cell (ASC) function.
|Priming Regimen||In Vitro Effects||In Vivo Effects||Model/Condition||Reference|
T-cell suppression ↑
PBMC proliferation ↓
|IFN-γ||IDO ↑||T-cell infiltration ↓|
Treg induction ↑
|Obliterative bronchiolitis model|||
|TNF-α||IL-6 secretion ↑|
IL-8 secretion ↑
|EP cell homing ↑|
|Ischemic hind limb model|||
|IFN-γ/ TNF-α||PGE2 ↑|
|IFN-γ/ TNF-α IL-17||iNOS ↑||T-cell suppression ↑||ConA-induced hepatitis model|||
|IFN-γ/ TNF-α/ IL-6||IDO ↑|
Cell diameter ↑
PBMC proliferation ↓
|IL-1β/ IL-6/ IL-23||No morphologic change|
CD45 expression ↑
Allogeneic T-cell proliferation ↓
TGF-β ↑, IL-4 ↓
|TGF-β||CXCR4 ↑||Cancer homing ↑|
Tumor volume ↓
Prolonged survival time ↑
|LPS||IL-6, TNF-α, HGF ↑|
|Liver regeneration ↑|
Serum AST, ALT ↓
|Partial hepatectomy model|||
IDO activity ↑
Restoring ASC inhibitory effect on pre-stimulated T cells
3.2.1. Genetic Modification to Enhance Retention and Migration
Since transplanted cells are vulnerable to the harsh microenvironment faced, most cells are cleared or turned to be dysfunctional in a short period of time, thus hindering their migration to the target site and them exerting their function. Therefore, prolonging the retention and improving migration capability are particularly important to improving the therapeutic efficiency of ASCs. The integration of the genes responsible for anti-apoptosis, self-renewal, and homing can be a favorable tool to achieve this goal. In addition, several efforts were made to increase the expression of multi-lineage differentiation and immunomodulatory genes in ASCs.
Sox2 and Oct4 are transcription factors that contribute to the maintenance of pluripotency in embryonic stem cells, as well as reprogram somatic cells into induced pluripotent stem cells. The forced expression of these two stemness-related genes was reported to enhance proliferation and prevent cell senescence in ASCs. Han et al. showed that ASCs overexpressing Sox2 and Oct4 exhibited enhanced proliferation, as well as osteogenic and adipogenic differentiation potential, in vitro by assigning ASCs a more primitive status [112
]. Moreover, other studies achieved the overexpression of both genes in a vector-independent way, via a combination of leukemia inhibitory factor (LIF) treatment and stem cell-specific miR-302 transfection, and found beneficial effects such as improved proliferation and reduced oxidant-induced cell death [113
]. In general, ASCs transduced with Sox2 and Oct4 showed remarkable benefits in their proliferation capability; however, it is important to note that more attention needs to be paid to a few conflicting results regarding differentiation potential [115
] and possible adverse effects such as tumor formation for clinical applications.
Upon hypoxic pre-treatment (the hypoxic pre-treatment strategy is discussed in Section 3.3.2
.), increased expression of superoxide dismutase 2 (SOD2) confers resistance to hypoxic stress by eliminating excessive reactive oxygen species (ROS), suggesting that SOD2 can be a convincing target of the genetic modification approach to improve ASC survival. In fact, ASCs virally overexpressing SOD2 resulted in significant survival improvement compared to transfected wild-type ASCs in vitro and in an in vivo syngeneic mice transplantation model [116
]. In an obsess diabetic mouse model, mice receiving SOD2-overexpressed ASCs exhibited beneficial outcomes in reducing adiposity and improving glucose tolerance through anti-oxidative and anti-inflammatory effects [117
C–X–C chemokine receptor 4 (CXCR4) signaling in response to its specific ligand stromal-derived factor-1 (SDF-1) serves as one of the critical factors involved in cell migration [118
]. SDF-1 is vigorously elevated under inflammatory or ischemic conditions, attracting preexisting or delivered stem cells expressing CXCR4 to regenerate the damaged tissue. Therefore, overexpression of CXCR4 can enhance migration and mobilization of ASCs through activation of the SDF-1/CXCR4 signaling pathway. ASCs following lentiviral transduction of CXCR4 showed strengthend proliferative and anti-apoptotic properties, as well as increased migration capability in vitro, possibly via the Erk pathway. [119
]. The enhanced homing and engraftment by CXCR4 gene transduction was consistently demonstrated as a result of significant muscle tissue regeneration in a mouse limb ischemic model [120
]. Similarly, overexpression of granulocyte chemotactic protein-2 (GCP-2/CXCL6) in ASCs demonstrated beneficial effects on an experimental myocardial infarction model. GCP-2 is another chemokine that contributes to tissue homeostasis, tumorigenesis, and angiogenesis, and ASCs with genetically overexpressed GCP-2 promoted the proliferation and migration capabilities that contribute to improving heart function [121
3.2.2. Genetic Engineering to Improve Immunomodulation
Unique immune-regulatory capacities are the most promising characteristics for the development and application of ASCs as a “medicinal drug” and, on the same line as pre-conditioning with pro-inflammatory cytokines, genetic modification approaches were undertaken to directly or indirectly enhance the major immunomodulatory mediators of ASCs. Based on the strengthened immunosuppressive effects on various immune cells in vitro, the enhanced therapeutic or prophylactic advantages of genetically modified ASCs were verified in several in vivo inflammatory disease models.
Several studies described that the incorporation of anti-inflammatory genes such as IL-10
], and Foxp3
] could improve the therapeutic potential of MSCs. In addition, since each disease has its own pathogenic mechanism, a variety of other genes can be targeted to elicit a specific beneficial effect on the particular disease. Payne et al. showed that ASCs engineered to overexpress IL-4
exerted protective effects in mice with experimental autoimmune encephalomyelitis (EAE) [126
]. Mechanistically, EAE as a model for MS is an antigen-driven autoimmune model characterized by abnormal differentiation and activation of CD4+
T cells biased toward the T helper 1 (Th1) and Th17 subsets. In contrast, IL-4 secreted from Th2 cells may act as an anti-inflammatory mediator that alleviates excessive Th1/Th17-induced inflammation in this model. Overexpression of the IL-4
gene in ASCs resulted in a reduction of antigen-specific T-cell responses and a compensational shift from pro- to anti-inflammatory cytokine response when delivered at early stage. Other studies revealed that genetically modified ASCs attenuated autoimmune arthritis in mice. CTLA4Ig
-modified ASCs ameliorated collagen-induced arthritis by reducing serum type II collagen (CII) autoantibodies and increasing the ratio of Treg (CD4+
) cells versus Th17 cells [127
]. Park et al. showed that the transduction of receptor for advanced glycation end products (sRAGE) led to higher expression of immunomodulatory factors in ASCs, including IL-10, TGF-β, and IDO, and enhanced migration capability. Moreover, arthritic IL-1Ra-knockout mice receiving sRAGE-overexpressed ASCs exhibited marked remission of inflammatory arthritis by downregulating Th17 cells and reciprocally upregulating Treg cells [128
There were also several studies on the introduction of genes encoding relatively newly identified cytokines into ASCs and the subsequent improvement of immunomodulatory properties. Marinez-Gonzalez et al. modified ASCs overexpressing soluble IL-1 receptor-like-1 (sST2), a decoy receptor for IL-33, and observed more pronounced pulmonary inflammation suppression and intact alveolar architecture than in naïve ASCs in endotoxin-induced acute lung injury models [129
]. This dramatic effect was attributed to synergy with the increased expression of immunomodulatory molecules, such as IDO, TNF-α-stimulated gene-6 (TSG-6), and CXCR4, in response to the local inflammatory environment, along with further inhibitory effects on IL-33, TLR4, and IL-1β production resulting from sST2 overproduction. Additionally, ASCs overexpressing IL-35 exhibited higher suppressive effects on CD4+
T-cell proliferation and IL-17 secretion compared with non-transfected MSCs in an in vitro coculture setting [130
Although the beneficial function enhancement of genetically modified ASCs was demonstrated as summarized in Table 2
, several limitations remain in their clinical application. The application of replication-defective viral vectors, such as lenti- and adenoviruses, is closely associated with safety concerns including potential tumorigenicity, toxicity, and immunogenicity [131
]. Moreover, the long-term curative effects, particularly at an organ or systemic level, are yet to be fully addressed. Therefore, extensive systemic studies are necessary to accumulate substantial evidence for the clinical application of gene-manipulated ASCs, including (1) the development of advanced gene integration methods with safety and efficiency, and (2) the elucidation of systematic in vivo mechanisms of genetically modified ASCs.
3.2.3. Genetic Manipulation to Induce Lineage Transdifferentiation
In addition to the three mesenchymal lineages, ASCs can also undergo transdifferentiation toward non-mesenchymal cell lineages, including myogenic, cardiac, endothelial, and neuronal cells, in response to the lineage-specific inducer [35
], although there are somewhat controversial views on neural transdifferentiation (reviewed in Reference [133
]). Lineage conversion can be achieved in vitro through the exposure of ASCs to extrinsic signaling molecules or via the modification of culture conditions, such as using specific biomaterials. Alternatively, genetic manipulation integrating key transcriptional factors into ASCs may be a better way to induce stable and effective lineage transdifferentiation [134
]. Although there are still obstacles to be overcome such as the development of safe gene delivery methods and the selection of the most appropriate target gene, encouraging evidence was accumulated over MSCs from different sources. In this section, we summarize the approaches to genetic manipulation to induce lineage transdifferentiation via overexpression of transcriptional factors in ASCs.
In the case of cardiomyogenic lineage, the forced expression of Tbx20
, a critical transcription factor that contributes to heart development and cardiomyocyte regeneration, efficiently induced expression of cardiomyogenic differentiation markers on ASCs at 14 days after transduction both at the RNA and protein level [135
]. It might be necessary to evaluate the cardiomyogenic regenerative capacity of Tbx20-overexpressed ASCs in an ischemic heart disease animal model. To transdifferentiate ASCs toward a neural lineage, Tang et al. transduced the proneural transcription factor Neurogenin (Ngn2) into ASCs and evaluated the in vitro neural lineage differentiation capacity and in vivo functional recovery in rat spinal cord injury (SCI). Rats transplanted with Ngn2-transduced ASCs showed higher expression of the neuron-specific nuclear protein (NeuN) in the injured site and exhibited the most striking functional recovery of the hind limb [136
]. To enhance myogenic differentiation, Goudenege and colleagues transduced the key myogenic gene MyoD
into human multipotent adipose-derived stem cells (hMADS) and observed a marked myogenic differentiation capability in vitro. Importantly, local intramuscular injection of MyoD
-overexpressed hMADS cells into the cryoinjured Rag2−/−
immunosuppressed mice significantly improved muscle repair with the increase in hMADS-derived muscle fiber [137
]. Moreover, it was reported that ETS variant 2 (ETV2) overexpression in ASCs can generate functional and expandable ETV2-induced endothelial-like cells (EiECs), which is expected to be an alternative strategy to treat ischemic vascular disorders [138
3.4. Application of Extracellular Vesicles (EVs)
It was suggested that the communication between MSCs and neighboring cells is pivotal for demonstrating MSC-derived therapeutic actions. The intercellular interaction is achieved not only via direct cell-to-cell contact (juxtacrine signaling) but also via a paracrine mechanism. Recently, EVs were regarded as a key mediator of paracrine signaling within the extracellular space [199
]. EVs are membrane-derived, lipid bilayer vesicular structures with various physiological functions. EVs are classified into several subtypes including exosomes and microvesicles according to their size, biogenesis pathway, and route of secretion. It was noted that EVs can reflect the biological status of parent cells since they contain bioactive molecules such as microRNAs, lipids, growth factors, chemokines, and cytokines. In this respect, it would not be surprising that purified EVs from the culture supernatant of MSCs could yield similar advantages to cells in many pathologic conditions [200
]. Indeed, several reports showed that long-lasting therapeutic outcomes could still be observed despite the high clearance rate of MSCs after in vivo application [203
], implying that MSC-derived secretory, soluble factors might play an essential role in cell therapy. More importantly, although the relevance of MSC application for incurable and intractable diseases was proven in a number of pre-clinical and clinical findings, conventional cell-based therapy has several limitations that need to be considered for practical application. For example, the quality of MSCs during the overall procedure from cell isolation, to expansion, storage, and transfer should be tightly controlled to maintain reliable therapeutic efficacy. Safety concerns regarding the ectopic differentiation of introduced MSCs, as well as unintended long-term inhibition of the recipient immune system, are other key issues to be monitored. Therefore, cell-free EVs attracted significant attention as an alternative therapeutic option in recent years.
In general, MSC-derived EVs (MSC-EVs) are isolated from MSC culture media via a serial ultracentrifuge procedure, then applied to the treatment of a wide range of abnormal pathologic conditions in which MSCs proved to be effective. Recent advances in “omics” technologies enabled researchers to define therapeutic candidates among MSC-secreted paracrine factors [205
]. Since one of the most significant roles of EVs is to mediate the horizontal transfer of parent cell-derived signaling molecules to target cells [208
], MSC-derived beneficial molecules such as TGF-β1 [209
], IL-10 [210
], PGE2 [211
], NO [212
], and IDO [210
] could be delivered via EVs. Therefore, the therapeutic actions of MSC-EVs are largely dependent on their tissue regenerative and immunomodulatory capacity as MSCs (Table 4
Until now, the therapeutic potential of ASC-EVs was analyzed in a wide range of pathologic conditions. Notably, many studies focused on the evaluation the advantages of EVs for neurological disorders because the central nervous system is hard to target with conventional cell delivery methods due to the presence of a blood–brain barrier.
To mimic the specific neurodegenerative condition, researchers used in vitro or in vivo transgenic models. Farinazzo et al. briefly proved the neuroprotective effect of ASC-EVs for a neuroblastoma cell line and primary hippocampal neurons under oxidative stress [216
]. Authors also showed that ASC-EVs could activate oligodendrocyte progenitors to regenerate the damaged myelin sheath after the treatment of a demyelinating agent during ex vivo cerebellar slice culture, implying that EVs can interact with both neuronal and non-neuronal cells. Bonafede et al. transfected motor neuronal cell line NSC-34 with mutant superoxide dismutase (SOD1) transgenes as observed in familial amyotrophic lateral sclerosis (fALS)-affected neurons, then assessed the neuroprotective impact of ASC-EVs in vitro [217
]. In this model, exosomes could protect SOD1-mutated motor neurons against hydrogen peroxide-mediated necrosis in a dose-dependent manner as previously reported [216
]. Katsuda et al. suggested the advantage of ASC-EVs for Alzheimer’s disease (AD) [218
]. Since the most significant etiology of AD is extracellular plaque formation within the brain caused by the aberrant accumulation of amyloid-beta protein, resolution of cerebral plaque is the key therapeutic strategy for AD. Based on the fact that neprilysin, one of the intensively studied endopeptidases for amyloid-beta proteolysis, is highly expressed in ASCs compared to BM-MSCs, Katsuda and his colleagues investigated whether ASC-derived exosomes had an anti-AD effect. They confirmed that exosomes isolated from ASCs contain physiologically active neprilysin. In addition, both extracellular and intracellular amyloid-beta produced by the N2a neuroblastoma cell line was degraded upon ASC supernatant treatment, implying that ASC-EVs might transfer ASC-derived neprilysin to neural cells. Meanwhile, an inflammation-induced demyelinating disorder, multiple sclerosis (MS), could be another target disease for ASC-EV therapeutics. It was reported that intravenously injected ASC-EVs could improve motor function while preventing brain atrophy in the murine model of Theiler’s murine encephalomyelitis virus (TMEV) infection, in which progressive MS-like symptoms are reproduced [219
]. Of interest, neuro-inflammatory signs such as glial cell accumulation and overexpression of pro-inflammatory cytokines were diminished in the EV-treated group compared to the vehicle-treated group. In addition, neural stem cell activity in the subventricular zone was found to be increased upon ASC-EV treatment. The therapeutic benefits of ASC-EVs were also evaluated in another widely accepted animal model for MS, experimental autoimmune encephalomyelitis (EAE) [220
]. In this paper, EVs were administrated to EAE mice before or after the disease onset, then clinical and histopathological severity was scored. It was noted that ASC-EVs might successfully play preventive roles in the progress of behavioral defects and neuroinflammation; however, they failed to rescue already developed EAE symptoms. EVs seemed to reduce inflammation partially via preventing the CXCL12–VCAM-1 mediated T-lymphocyte activation in the affected spinal cord.
In addition, many studies demonstrated that ASC-EVs could exert broad immunomodulatory and tissue regenerative roles in other disease models. Indeed, ASC-EVs ameliorated the atopic dermatitis-like skin lesions of mice via reducing the infiltration of innate immune cells such as mast cells and eosinophils [222
]. The proliferation and activation of T lymphocytes, one of the important components of adaptive immunity, was suppressed in the presence of ACS-EVs in vitro [221
]. Others reported that ASC-EVs stimulated the wound repair process by regulating fibroblast migration and re-assembly of the extracellular matrix within the damaged lesion [223
]. ASC-EVs could also provide protection against ischemic injury both in vitro and in a myocardial infarction mice model via stimulating Wnt/β-catenin signaling [224
]. Moreover, the pro-angiogenic effect of ASC-EVs was demonstrated by Liang et al [225
]. In this study, authors found that the messenger RNA (mRNA) level of angiogenesis markers including Ang-1 and Flk1 was increased in human umbilical vein endothelial cells treated with ASC-EVs. During both the in vitro tube formation assay and in vivo Matrigel plug assay, ASC-EVs stimulated the formation of a vascular-like tubular structure within the matrix through microRNA-125a transfer, an upstream negative regulator of anti-angiogenic Notch signaling, to endothelial cells.
Of note, modification and engineering techniques as described above could be applied to ASCs to produce superior EVs with higher therapeutic potential compared to naïve EVs. One paper demonstrated that MSCs primed with inflammatory cytokines IFN-γ and TNF-α could produce more immunosuppressive EVs compared to control MSCs, and basal proliferation levels of both innate (natural killer cells) and adaptive (T and B lymphocyte) immune cells declined more effectively in the presence of primed MSC-EVs than control MSC-EVs [227
]. In another study, EVs isolated from hypoxic-cultured ASCs exerted greater protection on cardiotoxin-induced skeletal muscle damage via inducing the class-switch of macrophages from pro-inflammatory M1 type to immunomodulatory and regenerative M2 type [228
]. Since most of the ASC-derived secretory molecules can be found in EVs, genetic modification aimed at the overexpression of therapeutic factors is also one of the preferred strategies for obtaining high-quality EVs. This strategy has several advantages, not only to avoid safety issues caused by genetic modifications but also to specify the mode of action for stem cell therapy. Yu et al. reported that EVs isolated from GATA-4-overexpressing MSCs prevented myocardial ischemic damage both in vitro and in vivo because they contained a higher level of anti-apoptotic microRNA miR-19a than control MSCs [226
]. In other studies, microRNAs known to play beneficial roles in a specific pathologic condition were directly overexpressed in ASCs to produce microRNA-rich EVs. Lou et al. produced miR-122-rich ASC-EVs via ASC transfection with a miR-122 expression plasmid. Interestingly, the chemosensitivity of hepatocellular carcinoma (HCC) toward anticancer agent sorafenib was improved upon miR-122-rich ASC-EV administration compared to naïve ASC-EVs, implying their therapeutic potential as an anticancer agent [229
]. Qu et al. conducted a similar study to target liver fibrosis with ASC-EVs rich in miR-181-5p, which is known to regulate autophagy. Upon treatment of powerful fibrosis-inducing cytokine TGF-β, hepatic stellate cells (HSCs) started to proliferate actively and produced a profound amount of ECM. It is noted that miR-181-5p-rich ASC-EVs suppressed HSC activation via directly targeting proliferative STAT3 signaling, leading to autophagy. The anti-fibrotic role of miR-181-5p-rich EVs was also proven in a CCl4-induced liver fibrosis model, where inflammatory factors and liver injury markers declined after modified EV injection. Therefore, it would be worthy to apply various strategies known to enhance the potency of ASCs to harvest therapeutically superior ASC-EVs. In addition, EV engineering techniques to improve targeting and migration efficiency, as well as further optimization of the EV handling process (from harvest and quantification to storage), should precede the practical translation of ASC-EVs.