Effect Of Mesenchymal Stem Cells Transfusion on the Diabetic Peripheral Neuropathy Patients .
Condition: Diabetic Peripheral Neuropathy
MeSH terms: Peripheral Nervous System Diseases; Diabetic Neuropathies
Study type: Interventional

Summary: 
      A debilitating consequence of diabetes mellitus (DM) is neuropathy which globally affects
      between 20 -30% of diabetic patients and up to 50% in other studies. The incidence of
      diabetic neuropathy (DN) is estimated to be up to 45% for type 2 diabetic patients and 59%
      for type 1diabetic patients in USA.(DN) is the most common complication of DM.The
      pathophysiology of DN is promoted by several risk factors: micro vascular disease, neural
      hypoxia, and hyperglycemia-induced effects.At the molecular level, the primary cause of
      diabetic complications is known to be hyperglycemia, which disrupts cellular metabolism by
      the formation of reactive oxygen species (ROS).In the aspect of nerve functions, ROS
      formation increases neuron's susceptibility to damage. In addition, hyperglycemia impedes
      production of angiogenic and neurotrophic growth factors, which are necessary for normal
      function of neurons and glial cells and maintenance of vascular structure.No definitive
      disease-modifying treatments have been to reverse DN. The current treatment focuses on tight
      glycemic control which can reduce potential risk factors for further nerve damage and
      DN-associated pain management.In many studies, deficiency of neurotrophic factors and lack of
      vascular support have been regarded as key factors in the development DN.Mesenchymal stem
      cells (MSCs) are particularly attractive therapeutic agents because of their ability to
      self-renew, differentiate into multi lineage cell types, and locally secrete angiogenic
      cytokines, including basic fibroblast growth factor (bFGF) and vascular endothelial growth
      factor (VEGF) .These factors were reported to prompt neovascularization and have support for
      neural regeneration.It was plausible that MSCs may also be an effective therapeutic agent for
      the DN treatment through the paracrine effects of bFGF (Shibata et al., 2008) and VEGF and
      their potential to differentiate into neural cells such as astrocytes, oligodendrocytes , and
      Schwann cells.The adherent nature of MSCs makes them easy to expand in culture and an
      attractive candidate to use in cell therapy.Therefore, cell therapy has recently emerged as
      an attractive therapeutic strategy to meet the needs of both neurotrophic and vascular
      deficiencies of DN.Proper diagnosis of DN depends on the pattern of sensory loss, reflex
      test, electrodiagnostic studies, and imaging
    

Detailed description: 
      Objectives This study will be conducted to investigate the effects of Mesenchymal stem cells
      (MSC) transfusion on diabetic peripheral neuropathy in diabetic patient. (MSCs) have been
      reported to secrete various cytokines that exhibit angiogenic and neuro supportive effects.

      Study Design Experimental interventional study. phase II clinical trial

      Ethical committee approval (was it ethically approved by the department) Internal medicine
      department Yes

      Study Methods

      Population of study & disease condition (e.g women with hepatitis, ………) Diabetic
      patients(type I, type II) with documented peripheral neuropathy as determined by impaired
      nerve conduction

      Inclusion criteria:

      (Type I, type II) diabetic patients age range (18-45) years, with diabetic peripheral
      neuropathy proved by clinical assessment and nerve conduction who did not receive treatment
      for diabetic peripheral neuropathy.

      Exclusion criteria:

      Decompensated cardiac, renal or liver disease. Associated autoimmune diseases Associated
      endocrinal diseases Pregnancy, usage of contraceptive pills or steroids.

      Methodology in details The study will be conducted on patients with diabetic peripheral
      neuropathy collected from internal medicine department(inpatient and out patients, males and
      females)

      All subjects of this study will be submitted to the following :( preparatory visit before
      (MSCs) transfusion visit.)

        -  Thorough clinical assessment as a general assessment of the patient, also assessment of
           diabetes complications especially diabetic neuropathy. This is including full history
           and examinations (e.g., pain, sensory loss, ulcers, sensory level, etc….) this
           preparatory assessment will be done at internal medicine department.

        -  Venous blood will be drawn in the morning after an overnight fast in the pre (MSCs)
           transfusion visit.

      Plasma biochemical blood measurements will be determined by standard laboratory procedures in
      the central lab at clinical pathology department, Kasr Alaini hospital)

        -  Fasting blood glucose level, 2 hours postprandial.

        -  C-peptide.

        -  Hb A1C.

        -  Basic Fibroblast Growth Factor (bFGF), vascular endothelial growth factor (VEGF) by
           ELISA.

        -  Complete blood picture.

        -  Liver functions in the form of ALT, AST

        -  Kidney functions in the form of serum creatinine.

             -  fundus examination

             -  Nerve conduction study at neurophysiology unit.

             -  Bone marrow aspiration of about 90 ml under local anesthesia once at the first
                visit after preparatory visit mentioned before, the patient will be admitted for 12
                hours and will be monitored closely to avoid anaphylaxis (by steroids,
                anti-allergic treatment), if no complication, will be discharged.

      To avoid infection: During bone marrow aspiration, procedure will be done under complete
      aseptic precautions, placed in sterile tubes containing pre-servative-free heparin
      (Sigma-Aldrich, St. Louis, USA) Separation and processing of the sample will be done under
      good manufacture procedure (GMP): Bone Marrow Aspirate (BMA) will be withdrawn under good
      sterilization of the skin in an isolated area. Processing of the sample will be done in the
      laminar air flow; all supplies are disposable and sterile.

      Technique:

      Separation of mononuclear cells:

      The bone marrow aspirate will be diluted at a ratio of 6:1 with phosphate buffer saline (PBS)
      with 2 mM EDTA (30 ml BM aspirate+ 5 ml PBS/EDTA buffer). The MNCs will be separated under
      aseptic conditions using a Ficoll. Hypaque desity gradient (density 1.077, GibcoBRL, Grand
      Islan, NY, USA) by centrifugation at 1800 rpm for 20 min then the MNCs will be plated in 40
      ml alpha-modi-field Eagle's medium (αMEM), serum free media; mesencult(Mesenchymal stem cell
      culture),penicillin (100 U/ml),streptomycin(10 mg/ml),0.5 ml amphotericin B(all from Gibco
      BRL) and 10 ng/ml basic fibroblast growth factor (b-FGF) (R&D system, Minneapolis, MN) and
      will be incubated at 370 c in a humidified atmosphere containing 5% CO2 (Digirolamo et
      al.1999).after one day ,non adherent cells will be cultured in the presence of Mesenchymal
      media for 3 weeks changed every 1 week (cambrex Bioscience ,Nottingham, uk). After reaching
      80% confluence the MSCs will be placed in 10 ml saline and will be infused intravenously

      Flow cytometry Surface expression of MSCs using anti- (CD271, CD34, CD73, CD90, CD105, CD29)
      monoclonal antibodies (mAbs) will be analyzed using flow cytometry. MSCs (2X105 cells) will
      be suspended in PBS containing 1% BSA and will be stained with flurochrome -conjugated mAbs
      for 20 minutes on ice (anti-mouse mAanti-CD 271, CD34 CD73, CD90, CD105,CD29; BD Bioscience,
      MN, USA).flow cytometric analysis will be performed using a FACSCaliber (BD
      bioscience)equipped with cell Quest software.10000 cells will be passed in front of the laser
      for each sample. Each sample will be analyzed in duplicate. A cut off value at 20% will be
      set to categorize samples as positive.

      Mesenchymal stem cells will be identified by morphology and immunophenotyping in the central
      lab at clinical pathology department, Kasr Alaini hospital( stem cell lab).

      Mesenchymal stem cells transfusion slowly intravenous will be applied after these procedures
      for the patients after taking their approval and informed consent.

      Follow up 3 months after Mesenchymal stem cells transfusion by fasting blood glucose level, 2
      hours postprandial, C-peptide, Hb A1C, (bFGF), (VEGF) and nerve conduction at kasr Alaini
      hospital departments as mentioned before.

      Possible Risk (mention if there is any risk or not) Anaphylaxis Infection

      Primary outcomes (Most important outcomes to be assessed)

      1- Effect of mesenchymal stem cells transfusion on diabetic peripheral neuropathy regarding
      improvement of clinical symptoms like pain, sensory loss and improvement of nerve conduction.

      Secondary outcome parameters (other outcomes to be assessed)

        1. Mesenchymal stem cells being a treatment modality in diabetes complications like
           peripheral neuropathy, to avoid hazards on patients secondary to diabetic peripheral
           neuropathy.

        2. Improving of diabetic condition like lowering of blood glucose level, decrease in
           insulin requirements and improvement of general condition.

      Sample size (number of participants included) 10 diabetic patients with diabetic peripheral
      neuropathy .

      Source of funding (is there any source of funds or not) Faculty Of Medicine, Cairo
      University.

      Time plan (when to start/ when expected to finish/ when to publish) At least 20 months
    

Intervention name(s): mesenchymal stem cells
Intervention type(s): Genetic
Intervention description(s): collection of stem cells by bone marrow biopsy from iliac crest, then culture for 1 month , then IV transfusion on 2 sessions to the same patient
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: diabetic peripheral neuropathy; mesenchymal stem cells