Allogeneic mesenchymal Stromal cells for Angiogenesis and neovascularization in no-option Ischemic Limbs

Severe limb ischemia (SLI) has major impact on quality of life, morbidity and
mortality. Many patients are not eligible for revascularization, leaving
amputation as only option. Mesenchymal stem or stromal cells (MSC), because of
their immunomodulatory and vasculoregenerative properties, may provide a novel
therapy for SLI. Allogeneic MSC therapy is attractive as it may be used as *off-
the-shelf* available treatment and allows testing and selection of isolates
before administration.

Primary Objective: To investigate whether intramuscular administration of
allogeneic MSCs is safe and potentially effective
Secondary Objective: To investigate the potential of various markers related to
inflammation, angiogenesis, and neovascularization to predict therapeutic
efficacy and prognosis.

A randomized, double-blinded, placebo-controlled clinical trial with 6 months
follow-up. Randomization will be performed by the Cell Therapy Facility of the
UMC Utrecht by means of a computerized table. Subjects will be allocated to
receive either MSC or placebo in a 1:1 fashion. The treatment (MSC) or placebo
will be administered by intramuscular injections in the most affected leg.

Intramuscular allogeneic BM-MSC injection: MSCs will be extracted from BM of
healthy volunteers, expanded with human platelet lysate, and stored. Patients
will be randomized (1:1) to receive intramuscular injections (30 sites) of
either placebo, or 150*106 allogeneic BM-MSC in the most affected limb. Blinded
syringes are provided and cell suspensions will be injected intramuscularly by
an experienced operator into multiple sites (30 sites, 1-1.5cm in depth, volume
of 1.0mL placebo or MSC per site) in the ischemic lower extremity.

Burden: Subjects will be screened during an inclusion visit (t=0). They will be
physically examined and blood samples will be taken (40mL). Baseline
measurements will be performed, including treadmill test, ABI, TBI, digital
photographs and measurements of any existing ulcers. An appointment for the
intervention will be made. There are 3 follow-up visits. At the first follow-up
visit, 1 week after the intervention, blood will be drawn and patients will be
screened for potential side-effects. Subsequent visits will take place at 2 and
6 months follow-up and include physical examination, treadmill test, ABI, TBI,
digital photographs and measurements of any existing ulcers. Quality of life
questionnaires (EuroQoL 5-D (EQ5D) and SF-36) will be administered at baseline
and 2 and 6 months follow-up. Patients are subjected to venepuncture four times
during which blood will be drawn (40 mL at inclusion and after 1 week and 20 mL
at each subsequent follow-up visit). After completion of the trial patients
will be contacted every year for 5 years to assess status of hard clinical
outcomes during long-term follow-up, i.e. limb status and mortality. In case of
death, the general practitioner will be contacted. Patients will also be asked
to fill out the quality of life (EuroQoL 5-D (EQ5D) and SF-36) questionnaires
every year, until 5 years after completion of the trial.

Safety: An independent Data and Safety Monitoring Board (DSMB) will review the
status and conduct of the clinical trial, evaluate all causes of death and
cardiovascular events and make recommendations to the clinical research group
concerning the trial*s continuation and modification. If in interim analyses
important clinical differences between groups become evident, the trial will be
stopped and patients will be offered the best treatment available. All patients
will be carefully monitored for side effects due to intramuscular injections,
changes in clinical parameters (temperature, blood pressure, heart rate) and
changes in renal, hepatic and metabolic parameters.