New Twist in Cell Therapy for the Treatment of Severe Ischemia

Peripheral vascular disease and critical limb ischemia remain major clinical problems worldwide. Decreased blood flow due to atherosclerotic occlusions and endothelial dysfunction are the most common pathogenic mechanisms in lower limb ischemia. For patients, quality of life is severely reduced due to low mobility, significant rest pain, and ulcerations that often lead to amputation. Current treatments are based on endovascular operations and bypass surgery, but they are not always effective due to advanced and diffuse disease and postoperative complications. Frequent comorbidities also affect the outcome of the current treatments. Thus, there is a clear need to develop new therapeutic approaches for millions of patients suffering from peripheral vascular disease.

During the last 20 years, critical limb ischemia has been the target of several gene and cell therapy approaches but unfortunately with no convincing clinical success.^1–5 Advanced disease and comorbidities have been considered as the main reasons for poor clinical results. Among tested cell therapy approaches have been intra-arterial and intramuscular injections of several types of adult stem cell populations, such as circulating endothelial progenitor cells and bone marrow and adipose-derived stem cells, but the results at best have been very modest and often attributed to paracrine effects from factors released by the administered cells rather than regeneration of functional blood vessels capable of increasing blood perfusion in the ischemic limbs.¹

In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Zhao et al⁶ report a new approach for vascular stem/progenitor cell (VSPC) therapy to induce neovascularization and rescue of ischemic hind limbs in mice. They use an elaborate Rainbow reporter mouse model and transplantation of cell sorting–isolated angiogenic mesenchymal stromal cells (MSCs) to show vessel-forming capacity of specific MSC subfractions in mice (Figure). They show that transplantation of VSPC 1 (CD45⁻Ter119⁻Tie2⁺PDGFRa⁻CD31⁺CD105^highSca1^low) and VSPC 2 (CD45⁻Ter119⁻Tie2⁺PDGFRa⁺CD31⁻CD105^lowSca1^high) subfractions alone did not lead to a meaningful outcome, but when the VSPC 1 and VSPC 2 subfractions were transplanted together, they had a synergistic therapeutic effect, induced functional neovascularization, and improved perfusion in the ischemic hind limbs. Importantly, similar functionally homologous subtypes of MSCs could also be isolated from human adipose tissue, thus paving a way for translation to the clinics.

The results reported by Zhao et al are encouraging and clearly move the cell therapy field forward. MSCs have been used for cell therapy previously, but the outcomes have been variable, and depending on conditions, these cells can also give rise to nonvascular cell types. It has now been recognized that MSCs are not a single multipotent population of cells but instead comprise several subtypes that can give rise to adipose, osteoid, myogenic, and endothelial progeny. Thus, accurate identification of cell markers used for the isolation of lineage-committed subsets, such as VSPC 1 and VSPC 2, is important for further development of MSC-based cell therapies. In the current work, multicolor lineage tracing in the Rainbow mice combined with fluorescent cell sorting and single-cell sequencing greatly helped to identify these subpopulations. This mouse model allows for an unbiased tracking of clonal expansion and vasculogenic activity by all potential cell types based on 10 possible color combinations of 4 fluorescent proteins cloned in the ROSA locus going beyond what can be detected by using lineage-restricted promoters.⁷ It should be noted that unlike induced pluripotent stem cell–based approaches, in which therapeutic cell fractions require prolonged in vitro culture to achieve expansion and differentiation, the approach used by Zhao et al is more rapid and, therefore, more feasible in a clinical setting.

An unexpected finding was that coadministration of VSPC 1 and VSPC 2 subfractions was required for a successful outcome. It appears that there is signaling cross talk between these 2 subfractions, which favors formation of functional blood vessels and inhibits differentiation into other lineages, such as adipocytes. These studies also confirm the importance of Tie2 signaling and Sca1 for the generation of functional vasculature in vivo. However, the exact mechanism behind the benefit of the coadministration still remains to be determined.

The approach described by Zhao et al mostly induces angiogenesis, but from the clinical perspective, it is likely that arteriogenesis (ie, enlargement of the preexisting small arterial conduits) is also needed to improve the overall condition of the ischemic legs because in the clinical setting, only large conduits can bring enough blood flow to relieve peripheral ischemia.¹ Thus, this new cell therapy approach would probably be most useful if combined with endovascular or open surgical operations. Also, apart from inducing new capillaries, their approach might help in a common clinical situation, in which the outcome of a technically successful endovascular or surgical procedure remains poor because the reperfused distal capillary network cannot accommodate the increased blood flow. This clinical scenario, termed poor runoff syndrome, often leads to thrombosis, as well as diversion of blood flow to vascular beds with the least resistance, leaving the most ischemic tissues with low or even no perfusion.

Obviously, mouse models are not optimal for testing the abovementioned conditions, and larger animal models are needed for the next steps in the clinical translation. Also, currently used mouse models do not have comorbidities like type 2 diabetes and hypercholesterolemia, which need to be addressed in the future since they will contribute to the microvascular dysfunction frequently present in patients with critical limb ischemia.

Since critical limb ischemia is a significant health problem worldwide, it is important to develop new therapeutic approaches. While exact mechanistic aspects, feasibility, dosing, and regulatory issues of the coadministration of the 2 MSC-derived subfractions remain to be clarified, this new cell therapy approach gives new hope for the development of effective cell therapy for severely affected patients who do not benefit from current endovascular or open surgical approaches.