Several studies have demonstrated that ABMMNC treatment for atherosclerotic peripheral arterial disease is a safe and effective strategy that achieves therapeutic angiogenesis and prevents amputation [17, 18]. In patients with TAO, several studies have reported that stem cell therapy induces development of new collateral vessels and improves ischemic symptoms in short-term studies [19, 20]. Our data demonstrate that stem cell therapy in patients with TAO has a 10-year amputation-free survival rate of 85.3%, with all five amputations being minor. In addition, improvements in ulcer area, TBI, TcPO2, and pain score remained significantly improved from the baseline and were significantly better than in the conservatively treated group, even after 10 years. Furthermore, there were no cases showing development of retinopathy or cancer. These data show the long-term safety and efficacy of ABMMNC therapy in patients with TAO. However, especially given the young age of these patients, strict life-time surveillance is required after ABMMNC therapy.

Previously published studies have suggested rare mortality or morbidity in patients with TAO who were treated with ABMMNCs [1820]. Consistent with these previous reports, our data similarly showed no deaths or severe adverse events. It is likely that the young age and few cardiovascular risk factors in patients with TAO may contribute to the safety of ABMMNC treatment. Miyamoto et al. [13] reported one death in a patient who was 30 years of age at 20 months after successful ABMMNC treatment; as no autopsy was performed, the cause of this single reported death after ABMMNC treatment remains unknown. Other than this single mortality, most studies have shown that cell therapy in patients with TAO is associated with few severe adverse effects. Therefore, in assessing stem cell therapy in patients with TAO, we believe that mortality is not a good indicator of treatment safety or efficacy.

Matoba et al. demonstrated that angiogenic cell therapy using bone marrow-derived mononuclear cells is associated with 3-year improvement in limb ischemia, leading to an extension of the amputation-free interval and improved perfusion compared to patients with atherosclerosis [21]. Our data are the first published 10-year results of ABMMNC treatment in patients with TAO and show the long-term safety and efficacy of this treatment. Interestingly, most outcomes, such as ulcer healing, toe pressure, TBI, and TcPO2, were improved in the first few months after treatment and then maintained during long-term follow-up (Table 2 and Fig. 3); this observation suggests that future studies can use shorter observation periods to determine the effects of treatment as well as adverse events. We believe that long-term improvement of symptoms and perfusion parameters with ABMMNC treatment may be due to three factors. First, patients with TAO have fewer cardiovascular risk factors; unlike patients with atherosclerotic limb ischemia, patients with TAO rarely showed involvement of their coronary, carotid, and visceral vessels, potentially reducing their risk for myocardial infarction, stroke, or mesenteric ischemia, respectively. Secondly, smoking and tobacco consumption are major factors associated with the development of TAO; since tobacco may trigger an immune response or unmask a clotting defect, tobacco can incite an inflammatory reaction in the vessel wall [22]. All patients that participated in this study signed a stop-smoking agreement. Interestingly, the modest clinical improvements in patients that stopped smoking but were not treated with ABMMNCs shows that smoking cessation alone may not improve ischemia, but it is likely that smoking cessation is a critical factor enabling proper stem cell function. Thirdly, lower extremity exercise training contributes to improved maximum walking distance, peak oxygen uptake, and quality of life in patients with intermittent claudication [23]. Since most patients with TAO rarely had severe comorbidities and generally had better physical condition than typical patients with atherosclerosis, it is likely that the patients who were treated with ABMMNCs were able to tolerate exercise training, giving additional benefit.

Of all the parameters that we evaluated, only the ABI showed no significant change after ABMMNC treatment, although the mean ABI increased slightly compared to baseline (Table 2 and Fig. 3). These results are similar to those reported in other studies; for example, in the Therapeutic Angiogenesis by Cell Transplantation (TACT) study [21], ABMMNC treatment showed no improvement in ABI after 3 years. It is likely that the lack of ABI improvement during long-term follow-up is related to the distal small arterial thrombosis that characterizes TAO [24]. Therefore, to evaluate long-term therapeutic results in patients with TAO, toe pressure, TBI, or TcPO2 may be more suitable outcome measures rather than ABI alone.

The mechanisms underlying stem cell therapy are still largely unknown. Yamamoto et al. reported that endothelial progenitor cells isolated from patients with TAO displayed high expression of endothelial lineage molecules compared with their counterparts obtained from patients with atherosclerosis [25]. This suggests that ABMMNC therapy can deliver bone marrow-derived endothelial progenitor cells that can incorporate into the existing vasculature, increasing capillary density and angiogenesis via paracrine effects [26]. It is also possible that delivery of ABMMNCs into the lower extremity hypoxic environment enhances their survival and/or function, as hypoxia promotes expansion of stem and precursor cell populations [27, 28].