Patients afflicted with CLTI have a poor quality of life and a high rate of limb loss [16]. Until now, revascularization of the ischemic limb, either by endovascular or open surgical approaches, has been the mainstay of therapy. However, 25–40% of people with CLTI are not suitable for it or have failed previous revascularization therapy, and the mortality rate remains high [17] [18] [19]; therefore, less invasive medical therapies that be effective in the treatment of CLTI are desirable.
In this context, new strategies such as regenerative medicine have enabled the development of therapeutic angiogenesis through recombinant proteins, gene transfer, or stem cells [5]. Nevertheless, the trials that only use recombinant proteins (growth factors) cannot provide the essential factors that patients with CLTI require. In the case of gene therapy, there may be relevant risks such as an increasing neo-vascularization in undesired tissue, malignant cell transformation and inflammation. Additionally, increased angiogenesis can destabilize atherosclerotic plaques, leading to arterial thrombosis. In particular, phase II and III clinical trials on angiogenic gene therapy showed mixed outcomes of positive and negative final results; thus, the role of gene therapy in vascular occlusive disease prevails unresolved [5][20]. According to this, when comparing the approaches based on proteins or genes vs cell-based therapies, the last ones are more beneficial because of their natural vasculogenic features and their paracrine impact [1].
Initial preclinical and small pilot clinical studies have demonstrated promising effects of cell therapy in PAD and CLTI. Particularly, the results of these studies suggest that most stem cell therapies can increase blood flow at the transplantation site by promoting angiogenesis and neovascularization through a direct effect of the administered cells on the vasculature, or by secretion of pro-angiogenic factors and modulation of the local immune response, which prevents amputation in patients with CLTI [12]. However, it is unclear whether BM-MNC or MSCs are more effective in both PAD and CLTI. In fact, some clinical trials gave inconsistent results revealing that BM-MNC are ineffective and MSCs may be superior [12] [18]. Based on this, the present work compared the effect of auto-BM-MNC vs. allo-WJ-MSCs in patients with CLTI. To our knowledge, this is the first study conducted in Colombia that compared the security and therapeutic potential of both cell-based therapies in diabetic patients with CLTI.
A crucial concern with stem cell therapy is its safety profile. Nevertheless, most of the published preclinical and clinical trials have reported that stem cells are safe to treat numerous injuries and diseases, including lower extremity vascular disease [21] [22].
The present study did not detect serious AEs, like malignancy, infection, organ system complications, or acute toxicity related to auto-BM-MNC or allo-WJ-MSC injections used to treat CLTI. In contrast, the participants receiving the placebo solution presented less ulcer healing and higher amputation rates. These findings were consistent with the evidence from numerous clinical trials that evaluated the safety of BM-MNC or MSC-based therapy in CLTI [2, 23, 24].
Regarding the effectiveness profile of the cell-based treatments, few clinical studies have aimed to compare the efficacy of different stem cells in treating CLTI in diabetic patients.
Some studies have reported that diabetic patients showed a marked depletion of CD34+ and a profound functional impairment of cultured EPCs [10] [9, 11]. These data suggest that the reduced number and dysfunction of EPCs in diabetes mirror an insufficient endogenous regenerative capacity, which favors the development of vascular complications.
Although our results strongly indicate that auto-BM-MNC and allo-WJ-MSCs increase limb’s blood flow and improve claudication symptoms of limb ischemia, we observe more promising results with allo-WJ-MSCs than auto-BM-MNC. Remarkably, in the clinical parameters that were evaluated, we observed an improvement in Rutherford’s classification, a significant increase in TcPO2 (values > 30 mm Hg), enhanced chronic ischemic ulcer healing, relief from a PWD, and increased amputation-free survival rates, which correlated with a recovery of the blood supply. In contrast, in the placebo group, the participants displayed more significant/higher amputation and surgical revascularization rates.
On the other hand, a number of preclinical and clinical studies have not demonstrated the clear benefits of auto-BM-MNC in various cardiovascular diseases [25]. Direct effects of cell aging on tissue repair capabilities are one of the strongest predictors of a lack of clinical response to auto-BM-MNC therapy; for instance, aged ECs are not only less effective at migration, but also more prone to become senescent, and have an altered secretion profile that contributes to the development of vascular complications [25].
Furthermore, the expected therapeutic angiogenesis using autologous BM-derived stem cells displays several disadvantages. Among them are the considerably limited amount of bone marrow obtained, and the procedure is painful for the patients. Besides, it may require general anesthesia, which can be life-threatening for patients with CLTI, who are already at elevated risk for difficulties due to their advanced age and cardiovascular disease. Similarly, the migration of circulating BM-derived stem cells is inefficient and significantly lower in patients with CLTI than in healthy subjects due to extended pro-inflammatory stimuli [20].
Recently, different studies have shown that autologous BM-MSC transplantation in patients with CLTI may have a risk of presenting karyotypic aberrations. Nonetheless, it is still not entirely understood if these abnormalities are innate of patients’ cells or have been originated during cell culture [26]. In this context, several studies have demonstrated that autologous MSCs obtained from patients suffering from inflammatory or degenerative diseases have variability in their biological and functional properties, provoking deleterious consequences for the host when dealing with host signals [27, 28]. Mainly, MSCs derived from individuals with atherosclerosis develop a pro-inflammatory secretome by the production of inflammatory cytokines such as IL6, IL8, and MCP1, reversing their naturally immunosuppressive properties [29]. Thus, the allogeneic cells allow for the best approach in cell-based therapy.
In this regard, studies that used Buerger's disease and ischemic limb disease animal models, demonstrated that umbilical cord blood-derived MSCs (UCB-MSCs) regenerated arterioles and promoted the differentiation into, UCB-MSCs offer various advantages due to (i) the newborn cell immaturity compared to adult stem cells and (ii) the ability to prevent immune reactions. In addition, the UCB-MSCs are less vulnerable to the attack of the recipient’s body than BM-derived stem cells. Several clinical trials have demonstrated that intramuscular administration of UCB-MSCs conduces to arterial reconstruction or prevention of arterial obstruction, decreases pain at rest and speeds up the healing process of ischemic ulcers. These studies proposed that growth factors or pain releasers secreted by implanted stem cells before vessel generation in ischemic regions, may be responsible for pain relief [30] [31].
Another clinical trial that used UCB-MSCs demonstrated the creation of new collateral arteries by computerized tomography angiography; this change was more apparent in the microvascular network than in the macrovascular network. Furthermore, compared to pre-treatment levels, the percentages of CD3+ CD8+ lymphocytes were significantly raised following treatment with UCB-MSCs, while percentages of CD3+ CD4+ lymphocytes and CD3-CD16/CD56+ NK cells were significantly reduced [20].
The MSC’s therapeutic role in patients with CLTI has been attributed to their unique biological features. MSCs can promote angiogenesis, reduce fibrosis, restore collagen balance, decrease immune cell activities, and undergo EC differentiation [32]. Likewise, MSCs and ECs engage in close cross-talk; specifically, MSCs stimulate the growth and relocation of ECs to initiate the early phases of angiogenesis, and lessen the permeability of the EC monolayer; this effect can be by both direct cell-cell contact and release of paracrine factors [33].
Different studies have shown that the MSC involvement in maintaining structures of neovessels in vivo is through several molecular pathways. Among those, the Wnt pathways are essential in adjusting MSC differentiation, proliferation, and migration. WNT4 activation in MSC increases blood flow. In particular, frizzled-related protein-1, a Wnt modulator secreted by MSCs, promotes angiogenesis by increasing MSC integration into neovessels, which implies that specific molecular targets are responsible for MSC engraftment into the vasculature, while TGF-β signaling regulates MSC differentiation into pericytes [34–36].
Regarding the best form of cell administration, most therapeutic trials addressing CLTI have focused on intramuscular cell administration as a more feasible and less harmful approach, demonstrating safety and effectiveness [37] [33]. However, in our study, auto-BM-MNC and allo-WJ-MSCs were administered into the periadventitial layer of the arterial walls; which generates a cell repository in situ.
Other administration routes are intravascular, which is more invasive and harmful since involves administering contrast materials that are injected into a vein, but is not recommended in patients with chronic renal disease [38]. Systemic delivery, such as intravenous (IV) or intra-arterial (IA) infusion, is used less frequently [39]. Notably, the IV route has shown many entrapments and lung embolus development [40]. Furthermore, the IV route inhibits EC proliferation and angiogenesis via cell-cell contact through modulation of the cadherin/catenin signaling pathways [41]. Other studies have reported thrombogenic events during IV MSC infusion [42, 43].
IA injection entails the danger of injury to the nerves and arteries, vessel wall dissection, and dislodgment of atherosclerotic plaques [37]. In our case, the periadventitial injection was easy to use, less invasive and demonstrated safety and effectiveness.
Another point of concern is the cell dosage to apply, which can vary according to the cell type/source [1]. Although the ideal number of cells to employ for angiogenesis is unknown, and there are limited studies on cell dose, in our study were administrated 15 injections of either BM-MNC (7.197x106 ± 2.984 x106 cells/mL each injection) or allo-WJ-MSCs (1.333 x106 cells/mL each injection), numbers that were safe and showed therapeutic benefit. Nevertheless, the administration of an excessive number of BM-MNC has resulted in adverse effects in animal models [20]. Therefore, dose studies will be critical to optimizing the administration of stem cell populations in CLTI.
On the other hand, the severity of ischemia in patients that are candidate for cellular treatment is a critical aspect to consider in clinical trials. In our study, the participants were in Rutherford’s stages 3–5, and responded effectively to the cellular treatment. This finding agrees with other studies, such as Walter et al., who reported that individuals with Rutherford’s stages 4–5 did respond to cellular treatment, but those with stage 6 did not [13].
We designed our study primarily as a pilot study and proof of concept, and even though the sample size was small, we saw a significant clinical improvement in the auto-BM-MNC and allo-WJ-MSC treated participants compared with the placebo group. Nevertheless, according to our research, allo-WJ-MSCs can rival auto-BM-MNC because of less invasive extraction approaches.
Finally, stem cell treatment may sometimes encounter ethical obstacles or biological restrictions. Thus, in recent years, it has been thought that the released secretomes, composed of biologically active molecules (growth factors, cytokines and chemokines, angiogenic factors, extracellular matrix proteins, proteases, and genetic material secreted from stem cells), have revealed a considerable capacity for repair and regeneration of damaged cell membranes, or induce the secretion of surrounding tissues could reveal a new approach for the cell-free treatment [44–46].