Mesenchymal Stem Cells in Patients With Type 1 Diabetes Mellitus
Condition: Type 1 Diabetes Mellitus
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 1
Study type: Interventional

Summary: 
      In this prospective, dual-center, open trial, patients with recent onset type 1 diabetes will
      receive one dose of allogenic Adipose tissue-derived stromal/stem cells (1x106 cells/kg) and
      oral cholecalciferol 2000UI/day for 24 months (group 1). They will be compare to patients
      that will receive just oral cholecalciferol 2000UI/day (group 2) and standard treatment
      (group 3: no treatment). Adverse events will be record. In addition, glycated hemoglobin,
      insulin dose, frequency of hypoglycemia, glycemic variability, % of time in hyper and
      hypoglycemia and peak response of the C-peptide after the mixed meal teste wil be measure at
      baseline (T0), after 3 (T3), 6 (T6), 12 (T12), 18 (T18), and 24 (T24) months.
    

Detailed description: 
      Adipose tissue samples will be obtain through liposuction procedures of three healthy
      volunteers undergoing aesthetic surgery at Clementino Fraga Filho (the hospital of Federal
      University of Rio de Janeiro, Brazil). Donors´sorology wil have to be negative for syphilis,
      Chagas disease, Hepatitis B virus, Hepatitis C, HIV 1 and 2, and Cytomegalovirus.

      The adipose tissue-derived stem/stromal cells (ASCs) that wil be extract on healthy
      volunteers will be isolate, culture and samples will be process at Core Cell Technology of
      Pontifícia Universidade Católica do Paraná. Briefly, the procedures are:

        1. 100 ml of adipose tissue will be wash in sterile phosphate-buffered saline (Gibco
           Invitrogen). A one-step digestion by 1 mg/ml collagenase type I (Invitrogen) will be
           performe for 30 minutes at 37°C during permanent shaking, follow by filtration step
           through a 100 µm mesh filter (BD FALCON, BD Biosciences Discovery Labware, Bedford, MA,
           USA). The cell suspension will be centrifuge at 800 g for 10 minutes, and erythrocytes
           were removed by lysis buffer, pH 7.3.

        2. The remaining cells will be wash at 400 g for 10 minutes and then culture at a density
           of 1×105 cells/cm2 in T75 culture flasks and DMEM-F12 (Gibco Invitrogen) supplemente
           with 10% of fetal calf serum, penicillin (100 units/ml) and streptomycin (100 μg/ml).
           The culture medium will be replace three days after seeding, and then twice a week.

        3. ASCs will be subculture after reaching 80% confluence, with 0.5% trypsin/EDTA
           (Invitrogen) solution. Cells will be replate at a density of 4x103 cells/cm2 for
           expansion11A.

        4. Quality control of cell suspension sterility will be evaluate by tests to detect
           bacteria and fungi (Bact / Alert 3D, Biomerieux), endotoxins (Endosafe ™ PTS, Charles
           River) and Mycoplasma (KIT MycoAlert ™ PLUS Mycoplasma Detection, Lonza). Cell viability
           will be performe by flow cytometry using the vital dye 7-AAD (7-Aminoactinomycin D -
           BD#559925) to determine the percentage of viable cells and Annexin V protein
           (BD#51-65875X) to determine the percentage of cells in apoptosis. Cytogenetic analysis
           wil be performe by GTG-banding method.

        5. Cells will be phenotypically characterize by flow cytometry before the clinical
           application, using the following monoclonal antibodies: FITC-labeled CD14 (BD#555397),
           CD45 (BD#555482), CD19 (BD#555412), CD44 (BD#555478); PE-labeled CD73 (BD#550257), CD90
           (BD#555596), CD166 (BD#559263), PerCP-labeled HLA-DR (BD#551375); APC-labeled CD34
           (BD#555824), CD105 (BD#562408), CD29 (BD#559883) all purchased from BD (Pharmingen). At
           least 100.000 events wil bel acquire on a BD FACSCalibur™ flow cytometer (BD
           Biosciences), and data wil be analyzed using FlowJo 10 (TreeStar) software11A.

      After the ASCs extraction, isolate, culture and process, the infusion in patients with
      recent-onset type 1 diabetes wil be according as describe bellow:

        1. At the day of infusion, ASCs monolayer wil be dissociate as described above and 1x106
           cells/kg of the recipient patient wil be ressuspend in 5 ml of saline solution with 50%
           albumin and 5% anticoagulant citrate dextrose solution. Cell suspension wil send to the
           hospital in a cooler with recycled ice.

        2. Patients that wil receive ASCs wil be admitte to the hospital in the day of the infusion
           and wil discharge 24 hours after infusion. A single dose of ASCs wil be infuse in a
           peripheral upper arm vein during 15-20 minutes. Patients wil start takking oral
           cholecalciferol 2000 UI one day after the ASCs infusion.

      Safety Tests: adverse events wil be record during the hospitalization (T0) and at each
      follow-up outpatient visit (3 [T3], 6 [T6], 12 [T12], 18 [T18], and 24 [T24] months after the
      ASCs infusion), with clinical and laboratory exams (blood count, lipids, renal and hepatic
      function, thyroid stimulating hormone, free tyroxine, antithyroglobulin antibody, calcium,
      phosphorus and 25-Hydroxy Vitamin D, performed with automated biochemical equipment CMD 800
      IX1).

      Clinical and Pancreatic Function Evaluation: Participants wil be followed for 24 months. On
      the first visit, all patients wil be interviewed and had a physical exam. Weight, height,
      body mass index (BMI), blood pressure, heart frequency, frequency of hypoglycemia and insulin
      dose/kg of body weight wil be evaluate in the first visit (T0) and after 1 (T1), 3 (T3), 6
      (T6) 12 (T12) ,18 (18), and 24 (24) months.

      Insulin dose adjustments wil be performe at each visit as necessary. All patients wil receive
      nutritional guidance according to American Diabetes Association recommendations.

      Blood samples wil be drawn prio to ASCs infusion and at T1, T3, T6, T12, T18 and T24 for the
      measurement the Glycated hemoglobin (HbA1c. Method: High Performance Liquid Chromatography by
      boronate affinity). β-cell function wil be evaluated through C-Peptide measurement
      (Microparticle Chemiluminescent Immunoassay method, Architect Abbott) after a liquid mixed
      meal (Glucerna®), considering the time 0 (basal), 30, 60, 90 and 120 minutes. The area under
      the curve wil be calculate.

      Comparison with previous case-control study using only vitamin D supplementation as
      intervention:

      The investigators wil compare our results with patients previously included in a case-control
      study that investigated the effects of daily 2000 UI vitamin D without the infusion of cells
      in individuals with recent onset type 1 diabetes and similar age (> 15 years old), from a
      different population (São Paulo) in the same country region (Brazilian Southeast). Therefore,
      the investigators wil establish three different groups for comparison: 1) patients that wil
      receive ASCs + Vitamin D supplementation; 2) patients that wil receive only vitamin D
      supplementation; 3) patients that wil receive the conventional treatment for diabetes but any
      additional experimental treament (ASCs or Vitamin D).

      Insulin dose adjustments or withdrawal wil be performed according to glycemic control.
      Changes in HbA1c, C-Peptide and insulin dose/kg of body weight wil be compare between groups.
      C-Peptide wil be analyze by immunofluorometric assay (AutoDelfia) at T0 and T6 and T12 and
      T18 and T24, considering basal and peak stimulated C-Peptide after mixed meal test
      (Glucerna).
    

Intervention name(s): Infusion of adipose tissue-derived stem/stromal cells and oral Cholecalciferol supplementation
Intervention type(s): Biological
Intervention description(s): The investigators will acess area under the curve of C-peptide after a liquid mixed meal (Glucerna®), considering the time 0 (basal), 30, 60, 90 and 120 minutes in each follow-up outpatient visit (3 [T3], 6 [T6], 12 [T12], 18 [T18], and 24 [T24] months after the adipose tissue-derived stem/stromal cells infusion). Other Clinical and Pancreatic Function Evaluation that wil be assess are: Weight, height, body mass index (BMI), blood pressure, heart frequency, frequency of hypoglycemia and insulin dose/kg of body weight, blood count, lipids, renal and hepatic function, thyroid stimulating hormone, free tyroxine, antithyroglobulin antibody, calcium, phosphorus and 25-Hydroxy Vitamin D.
Intervention MeSH terms: Cholecalciferol; Vitamin D
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Patients with recent onset type 1 diabetes wil receive an dose of allogenic adipose tissue-derived stem/stromal cells (ASCs) and oral Cholecalciferol UI/day for 24 months.
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: type 1 diabetes; pancreatic function; adipose tissue-derived stromal/stem cells