Allogeneic endometrial regenerative cells: an "Off the shelf solution" for critical limb ischemia?

Abstract

Critical limb ischemia (CLI) is an advanced form of peripheral artery disease which is responsible for approximately 100,000 amputations per year in the US. Trials to date have reported clinical improvement and reduced need for amputation in CLI patients receiving autologous bone marrow or mobilized peripheral blood stem cells for stimulation of angiogenesis. While such treatments are currently entering Phase III trials, practical and scientific pitfalls will limit widespread implementation if efficacy is proven. Hurdles to be overcome include: a) reduced angiogenic potential of autologous cells in aged patients with cardiovascular risk factors; b) invasiveness/adverse effects of bone marrow extraction and G-CSF mobilization, respectively; and c) need for on-site cellular manipulation. The Endometrial Regenerative Cell (ERC) is a mesenchymal-like stem cell derived from the menstrual blood that is believed to be associated with endometrial angiogenesis. We discuss the possibility of using allogeneic ERCs as an "off the shelf" treatment for CLI based on the following properties: a) High levels of growth factors and matrix metalloprotease production; b) Ability to inhibits inflammatory responses and lack of immunogenicity; and c) Expandability to great quantities without loss of differentiation ability or karyotypic abnormalities.

Figure 1

Stimulation of HUVEC proliferation by ERC conditioned media. ERC conditioned media was generated by 5 day culture of 10 × 10(5) cells in completed DMEM media. Conditioned media was added at the indicated concentrations to HUVEC cells in 96 well plates at a concentration of 5000 cells per well. Cells were incubated for 72 hours and proliferation as assessed by thymidine incorporation. Error bars represent ± Standard Deviation.

Figure 2

Limb preservation by ERC administration. 16 BALB/c female mice (6–8 weeks of age, Jackson Labs, Bar Harbor, Maine) underwent unilateral ligation of the femoral artery and its branches (superficial eplgastrlc artery) for induction of the limb ischemia. Additionally, ligation of N. peroneus for reproducing a neurotrophic ulcer-like injury was performed. Mice were divided into 2 groups of 8. Immediately after induction of injury, 1 million ERC were injected into the hind-limb muscle below the area of ligation. Cells were also injected on day 0, day 2 and day 4. ERC where injected in a volume of 200 microliters of saline. By day 14, necrosis was observed in legs of 8 control mice. 8 mice treated with ERC had intact limbs, with 2 displaying signs of impeded walking.

Figure 3

Immune modulation by ERC. A) In order to assess active proliferation of ongoing MLR 50,000 mitotically inactivated (mitomycin C) PBMC (stimulators) were incubated with 50,000 allogeneic PBMC (responders). Mitotically inactivated ERC were added at the indicated concentrations per well. Cells were cultured for 72 hours. For the last 18 hours of culture, cells were pulsed with 0.5 μCi 3H-thymidine. B&C) Ongoing MLR was established as described and supernatant was collected at48 hours. IFN-gamma and IL-4 were assessed by Quantikine Sandwich ELISA (R&D Systems, Minneapolis). D) Suppression of TNF-alpha production by ERC Conditioned Media. ERC conditioned media was added to mouse splenocytes that were activated with 2.5 microliters of LPS in a total volume of 200 microliters. The concentration of splenocytes was 250,000 cells per well. After culture for 48 hours, supernatant was examined for TNF-alpha by ELISA (R&D Systems). Error bars represent ± Standard Deviation.