Bone marrow cell transplantation has been shown to induce angiogenesis and thus improve ischemic artery disease. This study evaluates the effects of intramuscular bone marrow cell transplantation in patients with limb-threatening critical limb ischemia with a very high risk for major amputation. After failed or impossible operative and/or interventional revascularization and after unsuccessful maximum conservative therapy, 51 patients with impending major amputation due to severe critical limb ischemia had autologous bone marrow cells (BMC) transplanted into the ischemic leg. Patients 1-12 received Ficoll-isolated bone marrow mononuclear cells (total cell number 1.1 ± 1.1 × 10 9 ), patients 13-51 received point of care isolated bone marrow total nucleated cells (3.0 ± 1.7 × 10 9 ). Limb salvage was 59% at 6 months and 53% at last follow-up (mean 411 ± 261 days, range 175-1186). Perfusion measured with ankle-brachial index (ABI) and transcutaneous oxygen tension (tcpO 2 ) at baseline and after 6 months increased in patients with consecutive limb salvage (ABI 0.33 ± 0.18 to 0.46 ± 0.15, tcpO 2 12 ± 12 to 25 ± 15 mmHg) and did not change in patients eventually undergoing major amputation. No difference in clinical outcome between the isolation methods were seen. Clinically most important, patients with limb salvage improved from a mean Rutherford category of 4.9 at baseline to 3.3 at 6 months (p = 0.0001). Analgesics consumption was reduced by 62%. Total walking distance improved in nonamputees from zero to 40 m. Three severe periprocedural adverse events resolved without sequelae, and no unexpected long-term adverse events occurred. In no-option patients with endstage critical limb ischemia due to peripheral artery disease, bone marrow cell transplantation is a safe procedure that can improve leg perfusion sufficiently to reduce major amputations and permit durable limb salvage.

Although the immunomodulatory potency of mesenchymal stromal cells (MSC) is well established, the mechanisms behind are still not clear. The crosstalk between myeloid dendritic cells (mDC) and natural killer (NK) cells and especially NK cell-derived interferon-gamma (IFN-c) play a pivotal role in the development of type 1 helper (Th1) cell immune responses. While many studies explored the isolated impact of MSC on either in vitro generated DC, NK, or T cells, there are only few data available on the complex interplay between these cells. Here, we investigated the impact of MSC on the functionality of human mDC and the consequences for NK cell and Th1 priming in vitro and in vivo. In critical limb ischemia patients, who have been treated with allogeneic placenta-derived mesenchymal-like stromal cells (PLX-PAD), no in vivo priming of Th1 responses toward the major histocompatibility complex (MHC) mismatches could be detected. Further in vitro studies revealed that mDC reprogramming could play a central role for these effects. Following crosstalk with MSC, activated mDC acquired a tolerogenic phenotype characterized by reduced migration toward CCR7 ligand and impaired ability to stimulate NK cell-derived IFN-c production. These effects, which were strongly related to an altered interleukin (IL)212/IL-10 production by mDC, were accompanied by an effective prevention of Th1 priming in vivo. Our findings provide novel evidence for the regulation of Th1 priming by MSC via modulation of mDC and NK cell crosstalk and show that off-the-shelf produced MHCmismatched PLX-PAD can be used in patients without any sign of immunogenicity. STEM CELLS 2015;33:3087-3099 SIGNIFICANCE STATEMENTWhile MHC-mismatched mesenchymal stromal cells (MSC) are already used in clinical trials for tissue regeneration and treatment of immune-related disorders, these studies did not sufficiently address alloreactivity. In the present study, we provide to our knowledge for the first time immunological data from a clinical trial demonstrating that patients treated with MHCunmatched MSC did not develop a memory T cell response specific to the mismatch. Moreover, we demonstrate that MSC control important checkpoints of Th1 priming by inhibiting migration and cytokine production of myeloid dendritic cells resulting in a diminished ability to prime natural killer cells and Th1 cells.

The results of this first randomized placebo-controlled trial for autologous bone marrow cell therapy in CLI will clarify the value of this new therapeutic modality in a patient population with no other alternatives except major amputation.

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