Direct Evidence of Mesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments using In Vivo Bioluminescence Imaging methods

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    "name": "Direct Evidence of Mesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments using In Vivo Bioluminescence Imaging methods",
    "description": "Evidence-backed execution summary for Direct Evidence of Mesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments using In Vivo Bioluminescence Imaging methods from Direct Evidence of Mesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments using In Vivo Bioluminescence Imaging.",
    "totalTime": "PT23310M",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Materials and Methods",
        "text": "Human MSC (hMSC) were isolated from the bone marrow of normal individuals undergoing bone marrow harvest for allogeneic bone marrow transplantation following informed consent, according to institutional guidelines under the approved protocol, as described previously [ ]. Briefly, mononuclear cells were separated by centrifugation over a Ficoll-Hypaque gradient (Sigma, St. Louis, MO) and suspended in 10 ml of MSC complete medium: alpha-minimum essential medium (α-MEM) containing 20% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), L-glutamine and penicillin-streptomycin mixture (Gibco/Invitrogen, Carlsbad, CA) and plated in 180 cm 2 dish. After 3 days, the non-adherent cells were removed by washing with phosphate buffered saline (PBS) and monolayers of adherent cells were cultured until they reached confluence. Cells were then trypsinized (0.25% trypsin with 0.1% EDTA) and sub-..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Materials and Methods",
        "text": "Murine MSC (mMSC) were isolated as described previously [ ]. Briefly, femurs of 2-month-old Balb/C (Harlan Labs, ME) were collected, dissected into small fragment, then placed into a sterile mortar and crushed using a sterile pestle. Whole bone fragments and bone marrow pieces were then placed in toto into 10 ml MSC complete medium and plated in 180 cm 2 dish. After five days the plate was washed to remove non-adherent cells After two complete washings adherent cells were retrieved by trypsinization and immunodepleted of granulo-monocytic cells using a biotinylated antibody against CD11b (BD Biosciences, San Jose, CA), and streptavidin-coated microbeads from Miltenyi Biotec (Auburn, CA) according to the manufacturer's instructions. After immunodepletion, the remaining cells were plated in fresh media, and within 3 additional days, fibroblast-like colonies were observed. Medium was cha..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Materials and Methods",
        "text": "To determine the multi-lineage differentiation potential of the human MSC, we subjected passage 3 hMSC to various differentiation media, according to manufacturer recommendations (Miltenyi Biotec, Auburn, CA). Briefly, hMSC were cultured for 3 weeks in NH AdipoDiff, NH OsteoDiff, or ChondroDiff media with media changes ever 3 days. Adipocytes were then stained with Oil Red O (Sigma), and osteoblasts were stained for calcium with Alizarin Red S. For chondrogenic detection, hMSC pellets were sectioned and stained with Alcian Blue. ffLuc labeled MSC were also assayed for adiopgenic, osteogenic and chondrogenic potentials."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Flow Cytometry",
        "text": "MSC were harvested with 0.25% trypsin-EDTA and resuspended in phosphate buffered saline (PBS) supplemented with 2% FBS. Approximately 1 × 10 6 cells were stained with 1µg of antibody for 30 minutes at 4°C, and then analyzed on a FACSCaliber (Becton-Dickson, San Jose, CA). Human antibodies used included CD105, CD90, CD140b, CD73, CD166, CD44, CD146, CD31 and CD34 (BD Bioscience, San Jose, CA). Antibodies specific for mouse antigens included Sca-1, CD106, CD140b, CD44, CD31, CD11b and CD45 (eBiosciences, San Diego, CA)."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Adenoviral vector and MSC transduction",
        "text": "A recombinant adenoviral (Ad) vector expressing firefly luciferase (ffLuc), possessing an RGD-modified fiber (AdLuc-F/RGD) was prepared, purified, and titered as described previously [ ]. Human MSC were incubated with AdLuc-F/RGD at 3,000 viral particles per cell (vp/cell) (based on OD reading) for 4 hours, while Balb/C and C57Bl/6 MSC were incubated with AdLuc-F/RGD at 5,000 vp/cells for 6 hours in serum-free medium. After incubation serum containing media was added to the culture. Transduction efficiency was routinely above 95% in both hMSC and mMSC, as previously reported [ ]. MSC were assessed for luciferase expression by plating 5 × 10 4 transduced MSC into 24 well plates and adding 1µl of D-Luciferin (40mg/ml stock) (Caliper Life Sciences [Xenogen], Hopkinton, MA) into 2ml culture medium. 30 sec later cells were placed into the imager for detection. Using this multiplic..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "MSC administration to non-tumor bearing animals",
        "text": "1 × 10 6 hMSC-ffLuc suspended in 100µl PBS were injected into the tail vein of SCID mice (n = 5). The mice were imaged for bioluminescence detection to pinpoint hMSC-ffLuc location at 1, 3, 5, 7, and 10 days post MSC injection. Likewise, 1 × 10 6 mMSC-ffLuc generated from a Balb/C mouse, suspended in 100µl PBS were IV injected via tail vein in syngeneic Balb/C mice (n=5). These mice were imaged 1.5, 2.5, 10, 18, and 24 hours post mMSC-ffLuc injection."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Cutaneous wounding models and MSC administration",
        "text": "Anesthetized SCID mice were shaved and cleaned with sterile alcohol and gauze. A subcutaneous surgical incision (1.25cm) was made on the right side of the abdomen (n=3). This incision was immediately sutured shut with 5 stitches, and the mouse was isolated in a private cage. Three days post-surgery, the mice were injected via tail vein with 2.5 × 10 5 hMSC-ffLuc. The tail vein puncture wounding experiments were done on mice receiving 3 separate injections using a 27 gauge needle along the lateral tail veins during the injection of 2.5 × 10 5 ffLuc-MSC (n=5). All wounded mice were imaged over a period of 2 weeks, at which time point they were sacrificed."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Tumor models and MSC administration",
        "text": "Xenograft tumors: 5 × 10 5 MDA-MB-231 cells in 100µl of PBS were administered IV via the tail vein of SCID mice and allowed to seed the lungs (n=5). After 10 days, 1 × 10 6 ffLuc labeled hMSC (hMSC-ffLuc) were IV injected in 100µl PBS. hMSC-ffLuc localization was tracked by bioluminescence imaging twice per week until sacrifice of the animals at Day 29. 5 × 10 5 HEY ovarian carcinoma cells were suspended in 1 ml of PBS and intraperitoneally (IP) injected into SCID mice (n=3). 15 days post-tumor injection, 1 × 10 6 hMSC-ffLuc were IP injected in 100µl PBS, and these cells were monitored 3 times per week for 2 weeks, at which time point these animals were sacrificed. Syngeneic tumors: 2.5 × 10 5 Balb/C derived murine 4T1 breast carcinoma cells were subcutaneously injected into the hind limbs of Balb/C mice. After 10 days of tumor establishment, 1 × 10 6..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "Flow Cytometry"
      },
      {
        "@type": "HowToTool",
        "name": "Bioluminescent imaging"
      },
      {
        "@type": "HowToTool",
        "name": "Detection of MSC by immunohistochemistry and fluorescence"
      },
      {
        "@type": "HowToTool",
        "name": "Statistical Analysis"
      }
    ],
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        "@type": "HowToSupply",
        "name": "Materials and Methods"
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        "@type": "HowToSupply",
        "name": "Materials and Methods"
      },
      {
        "@type": "HowToSupply",
        "name": "Cell Culture"
      },
      {
        "@type": "HowToSupply",
        "name": "Flow Cytometry"
      },
      {
        "@type": "HowToSupply",
        "name": "Adenoviral vector and MSC transduction"
      },
      {
        "@type": "HowToSupply",
        "name": "Detection of MSC by immunohistochemistry and fluorescence"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Direct Evidence of Mesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments using In Vivo Bioluminescence Imaging",
      "datePublished": "2009",
      "author": [
        {
          "@type": "Person",
          "name": "Shannon Kidd"
        },
        {
          "@type": "Person",
          "name": "Erika Spaeth"
        },
        {
          "@type": "Person",
          "name": "Jennifer L. Dembinski"
        },
        {
          "@type": "Person",
          "name": "Martin Dietrich"
        },
        {
          "@type": "Person",
          "name": "Keri Watson"
        },
        {
          "@type": "Person",
          "name": "Ann Klopp"
        },
        {
          "@type": "Person",
          "name": "Lokesh Battula"
        },
        {
          "@type": "Person",
          "name": "Micheal Weil"
        },
        {
          "@type": "Person",
          "name": "Michael Andreeff"
        },
        {
          "@type": "Person",
          "name": "Frank C. Marini"
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      ],
      "identifier": "10.1002/stem.187"
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