Mesenchymal stem cells (MSCs) have a powerful immunosuppressive capacity, and they have been used to treat numerous immune diseases, such as refractory graft-versus-host disease. Nevertheless, there are conflicting clinical data. To our knowledge, MSCs from different donors do not share the same qualities and have different immunosuppressive capacities. Infused MSCs are cleared by the recipient’s immune cells or macrophages. Therefore, the MSC therapeutic strategy might be the most important factor that determines treatment success. Repeated infusions would lead to a relatively stable MSC concentration, which would benefit a sustained therapeutic effect. In this review, we focus on the quality of MSCs and the associated therapeutic strategy, as well as other potential variables affecting their utility as a cellular pharmaceutical.
Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established form of therapy for malignant hematological disorders [1,2]. The treatment is complicated by treatment-related mortality because of infections, regimen-related toxicity, and engraftment failure [3,4,5]. Graft-versus-host disease (GVHD) is a pivotal factor that contributes to engraftment failure [6,7], despite an intensive preparative regimen, an adequate cell dose, and complete donor chimerism. Patients with grade III and grade IV acute GVHD have estimated 5-year survival rates of 25% and 5%, respectively .
Because of their powerful immunosuppressive ability, mesenchymal stem cells (MSCs) have been used in the treatment of immune diseases. Animal studies have shown that MSCs improve the engraftment of HSCs and ameliorate GVHD [9,10,11,12], and subsequent clinical data have further confirmed their efficacy in treating refractory GVHD [13,14,15,16,17]. For example, patients receiving co-transplantation of MSCs at the time of cord blood transplantation (CBT) had a significantly faster hematopoietic recovery of neutrophils and platelets than those receiving CBT alone (P=0.02 and 0.01, respectively) .
However, negative results from a phase III trial on the use of MSCs to treat steroid-refractory acute GVHD almost ended this avenue of study . A review analyzed the possible reasons that contribute to four distinct MSC product variables: donor variance, epigenetic reprogramming, immunogenicity, and cryopreservation . Furthermore, another comprehensive study focused on discrepancies in clinical trials that used autologous MSCs to treat heart disease, finding that only trials containing flaws, such as in study design or reporting errors, showed positive outcomes [20,21]. Hence, investigators might need to identify the critical impacts of MSCs on therapeutic efficacy and optimize MSC therapeutic strategies according to different clinical indications. In this review, we focus on two pivotal parameters of MSC-based cytotherapy: quality of MSCs and therapeutic regimen (dose and frequency).
2 Quality of MSCs
Before addressing questions regarding the definition of, as well as the characteristics that are closely related to, the quality of MSCs, several characteristics that have been used to reflect the MSC quality, such as proliferative potential, the scale of cell senescence, and immunosuppression, will be discussed.
Aging, as well as long-term culture and diseases, exert a very important influence on the quality of MSCs. Several early investigations have provided evidence that the loss of differentiation potential and loss of proliferative potential are the most common age-related changes [22,23,24,25,26]. Age-related changes also include a decreased viable number of cells according to fibroblast colony-forming unit assay results and an increased number of senescent cells [26,27]. Furthermore, MSCs from aged individuals lose the ability to replenish progenitor cells [27,28].
After allogeneic HSCT, the frequency of bone marrow (BM)-MSCs within the BM-mononuclear cell (MNC) fraction was shown to be significantly reduced compared to healthy donors, which suggested that HSC transplantation may be the main reason for MSC damage [29,30]. BM-MSCs from hematological disease patients receiving high-dose chemotherapy display low levels of clonogenic potential and a defective proliferative capacity, indicating potential disadvantages of the use of autologous MSCs in chemotherapeutically pretreated patients . Furthermore, the number of MSCs is estimated to be small in an average BM harvest (2–5 MSCs per 1 × 106 MNCs, suggesting that a BM graft composed of 2 × 108 MNCs/kg contains 400–1000 MSCs/kg) . We have discussed and summarized the relation between abnormalities of self-MSCs and diseases . Better clinical results can be obtained using healthy allogeneic MSCs (off-self).
After expansion in vitro, MSCs inevitably undergo replicative senescence [34,35,36,37]. In later passages, the number of enlarged MSCs increases , and telomeres are shortened . Later-passage MSCs initiate significantly more instant blood-mediated inflammatory reactions  and activate the complement pathway [39,40], limiting their survival and function in vivo. It has been reported based on in vitro experiments that the immunosuppressive effects of MSCs did not differ from passage 2 to passage 7 . However, in therapy-resistant GVHD patients, the 1-year survival rate was 75% in patients who received healthy donors’ early-passage MSCs (from passages 1–2) in contrast to a rate of 21% when using later-passage MSCs (from passages 3–4) .
3 Therapeutic Strategy (Dose and Frequency)
When using MSCs to treat diseases, the dose may be the first important factor to consider. More evidence is needed to define the optimal dose. Patient conditions are more complicated than those of animal models. MSCs are not specific enough to exert an exclusive therapeutic effect on one condition, such as an immune reaction or a tissue injury. When an immune reaction and a tissue injury coexist, MSCs not only suppress the immune reaction but also repair the injured tissue. Therefore, the same dose might not be appropriate for different patients. The doses of MSCs commonly used in most clinical studies are listed in Table 1. However, questions such as what would happen when a higher dose of MSCs is used in a single infusion and whether there is a human maximum tolerated dose of MSCs remain unanswered.
Intervention characteristics of clinical studies on MSC-treated GVHD
|Published years||Study design||Age of patients||Status of GHVD||MSC source||MSC passage||Frequency||Dose/infusion||Response|
|2004||1 patient: case report||9 years||Severe, treatment-resistant, grade IV, acute||Allogeneic BM||N||2 doses, separated by 77 days||1st: 2.0 × 106/kg; 2nd: 1.0 × 106/kg||100%|
|2008||Multicenter, phase II study||0.5–64 years||Severe, steroid-resistant, acute, grades II–IV||Allogeneic BM||1–4||27 patients: 1 dose; 22 patients: 2 doses; 6 patients: 3–5 doses||0.4–9.0 × 106/kg||Complete: 53%; others: 16%|
|2009||Randomized, multicenter, phase II trial||34–67 years||Acute, grades II–IV||Allogeneic BM||5||1 dose||Two groups: 2.0 × 106/kg; 8.0 × 106/kg||Complete: 77%; partial: 16%|
|2011||Multicenter||0.4–15 years||Severe, steroid-refractory, acute, grades III–IV||Allogeneic BM||N||Twice weekly for 4 weeks||2.0 × 106/kg; 8.0 × 106/kg||Complete: 58%; partial: 17%; mix: 25%|
|2012||Single center||1–67 years||Steroid-refractory, acute||Allogeneic BM||1–4||10 patients: 2–5 doses||0.65–3.0 × 106/kg||Complete: children: 83%; adults: 44%; MSCs 1–2P: 86%; MSCs 3–4P: < 36%|
|2013||Two centers||0.7–18 years||Steroid-refractory, acute, grades III–IV||Allogeneic BM||2–3||22 patients: 2 doses; 12 patients: 3–5 doses; 3 patients: > 6 doses||0.9–3.0 × 106/kg||Complete: 65%; partial: 22%|
|2014||Open–label, multicenter||0.2–17.5 years||Severe, steroid-refractory, acute, grades II–IV||Allogeneic BM||N||1 stage: twice weekly for 4 weeks; 2 stage: once weekly for 4 weeks||2.0 × 106/kg||1 stage: complete: 61.3%; 2 stage: complete: 40%*|
|2016||Randomized, multicenter, double-blind controlled, phase II||N||Chronic||Allogeneic UC||N||1 dose per month for 2–4 months||3.0 × 107 per patient||Treatment: 72.6%; control: 51%|
Although one infusion of MSCs has been used in most clinical studies, increasing amounts of data suggest that multiple infusions could improve effectiveness. Studies have shown that <1% of transplanted cells survive longer than 1 week after injection for treatment of cardiac diseases [43,44], which means that multiple infusions are needed to maintain a therapeutic effect. This result is supported by animal experiments that showed that only multiple infusions of MSCs could prevent GVHD . There was no difference with respect to efficacy between the low and high Prochymal® (industrial MSC product) doses if a single treatment was used . Moreover, some GVHD patients needed repeated MSC infusions to obtain a better response [42,47,48]. Repeated infusions of MSCs were also shown to lead to better clinical outcomes in liver failure diseases [49,50], whereas a single treatment of MSCs failed to result in marked long-term (48 weeks) outcomes .
It is important to note that good clinical outcomes are obtained when MSCs are combined with drugs and/or other treatments, instead of therapy with MSCs alone. For example, MSC therapy for the treatment of acute GVHD reached 100% response with the addition of corticosteroid therapy .
4 Other Factors
The cell delivery route may have a critical effect on therapeutic efficacy for clinical translation. Most MSCs are trapped in the lungs after intravenous injection (>70%) . Compared to intravenous injection, the intraperitoneal injection of MSCs resulted in better colitis recovery in the treatment of mice with dextran sulfate sodium (DSS)-induced colitis . MSCs have the characteristic of chemotaxis, which enables their migration to sites of injured/inflammatory tissue. Preparing MSCs with optimal homing capacities might improve the efficacy of therapeutic applications .
It has been found that repeated infusions of MHC-mismatched MSCs from the same source leads to MSC alloimmunization and the subsequent rejection of MSCs by the host immune system [55,56,57]. These animal data suggest that using different sources is a better option for repeated infusions of MSCs.
Culturing MSCs in fetal calf/bovine serum containing xenoantigens may also enhance the intrinsic immunogenicity of MSCs [58,59]. The use of platelet lysate may also circumvent this possible problem . Additionally, freshly thawed cryopreserved MSCs have been shown to upregulate heat shock proteins, be refractory to interferon-γ, and cooperate in suppressing CD3/CD28-driven T-cell proliferation . Animal experiments have provided evidence that interferon-γ enhances the suppression function of MSCs [62,63,64]. However, following IFN-γ stimulation, MSCs induce the surface expression of de novo major histocompatibility complex (MHC) II  and present antigens as professional antigen-presenting cells [66,67]. Therefore, more clinical studies are needed to clarify whether MSCs require licensing by IFN-γ.
5 Discussion and Conclusion
Steroid-refractory GVHD is a devastating disease, and about half of the patients have a very poor overall survival rate of <10% [8,68]. Numerous animal data and results from small-scale clinical studies have demonstrated that promising outcomes can result from the powerful immunosuppressive capacity of MSCs. However, multiple transfusions of Prochymal failed to meet its primary clinical end point of achieving an increased GVHD overall complete response rate, compared with a placebo control, in a large-scale clinical trial . Intrinsic heterogeneity, coupled with large-scale clinical manufacturing, may explain, in part, why data from MSC-based clinical trials are largely incongruent . A meta-analysis review found that MSC treatment had a beneficial effect on the 6-month survival rate of patients with steroid-refractory acute GVHD and that survival did not differ with respect to age, MSC culture medium, or dose of MSCs delivered . However, another clinical study showed that the response rate was better among children than among adults treated for GVHD (83% complete or partial response, compared with 44% for adults), and this result was reflected in a significantly better survival rate .
GVHD is an immune disease caused by host T-cells attacking the tissues of the transplant recipient. MSCs have a powerful immunosuppressive capacity to suppress T-cell activation and proliferation, which should contribute to GVHD treatment. To the best of our knowledge, conflicting clinical outcomes may be the result of differences in experimental design. Therefore, careful attention should be paid to the quality of MSCs (intrinsic heterogeneity) and to the therapeutic strategy.
Conflict of Interest: The authors state no conflict of interest.
Authors’ Contributions: HY Wang made the literature analysis and wrote, discussed, and revised the manuscript of this review. WH Kuang critically analyzed and corrected the manuscript. All authors read and approved the final manuscript.
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40% patients achieved complete resolution of acute GVHD with additional 4 times MSC infusions when these patients failed to achieve complete response in the first stage treatment. BM = bone marrow; GVHD = graft-versus-host disease; MSC = mesenchymal stem cell; UC = umbilical cord.