Expanding Donor Muscle‐Derived Cells for Transplantation

Maura H. Parker1, Stephen J. Tapscott2

1 Department of Medicine, University of Washington, Seattle, Washington, 2 Department of Neurology, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2C.4
DOI:  10.1002/9780470151808.sc02c04s25
Online Posting Date:  May, 2013
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Abstract

Studies in mice showed tremendous promise for the eventual clinical utility of myoblast transplantation to treat human muscular dystrophies. Initial attempts to translate the murine studies to humans, however, were not successful, due in part to limited engraftability of expanded donor myoblasts. Conventionally, muscle cells have been cultured on collagen‐coated tissue culture‐treated polystyrene. However, this promotes lineage progression and differentiation of cells, which limits engraftment potential. This unit describes the isolation of canine muscle‐derived cells, ex vivo expansion of cells on plates coated with a modified Notch ligand, and the xenotransplant method used to evaluate engraftment potential. Activation of Notch signaling in freshly isolated canine muscle‐derived cells with Delta‐1ext‐IgG inhibits myogenic differentiation, and maintains cells earlier in myogenic lineage progression. Delta‐1ext‐IgG‐expanded cells engraft into the regenerating muscle of NOD/SCID mice more effectively than control cells expanded on human IgG, as evidenced by a significant increase in the number of muscle fibers expressing canine dystrophin in recipient murine muscle. Therefore, this protocol provides the basis for further developing culture conditions for ex vivo expansion of donor muscle cells for transplant. Curr. Protoc. Stem Cell Biol. 25:2C.4.1‐2C.4.16. © 2013 by John Wiley & Sons, Inc.

Keywords: Notch signaling; skeletal muscle; satellite cells; transplantation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Canine Muscle‐Derived Mononuclear Cells for Culture and Transplant
  • Alternate Protocol 1: Two‐Step Digestion Method to Increase the Proportion of Myogenic Cells
  • Support Protocol 1: Cell Counting Using a Hemacytometer and Setting up Dilutions for Culture
  • Support Protocol 2: Generating Delta‐1ext‐IgG and Human IgG‐Coated Plates
  • Support Protocol 3: Determining the Percentage of Myogenic Cells in the Freshly Isolated Cell Suspension by Counting Colonies
  • Basic Protocol 2: Transplantation of Expanded Cells
  • Support Protocol 4: Preparing Mice for Transplantation
  • Support Protocol 5: Analyzing Injected Muscle by Immunostaining of Cryosections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Canine Muscle‐Derived Mononuclear Cells for Culture and Transplant

  Materials
  • Skeletal muscle sample
  • Dulbecco's phosphate‐buffered saline (D‐PBS; calcium‐ and magnesium‐free)
  • Wet ice
  • Dulbecco's modified eagle's medium (DMEM), high glucose
  • Collagenase, Type 4 (Worthington Biochemical, cat. no. LS004186)
  • Growth medium (see recipe)
  • Plates coated with Delta‐1ext‐IgG, human IgG (see protocol 4)
  • 15‐ and 50‐ml conical polystyrene tubes
  • 10‐cm glass petri dishes, sterile
  • Microdissecting scissors and forceps
  • Paper towels, sterile
  • Two scalpel blade holders with no. 11 blades
  • Parafilm
  • Water bath set at 37°C
  • Plastic disposable Pasteur pipets
  • Pipet‐aid
  • 5‐ml plastic pipets
  • 20‐ml syringes
  • 16 1/2 and 18 1/2‐G needles
  • 70‐ and 40‐µm cell strainers [BD Biosciences, cat. no. 352340 (40 µm) and cat. no. 352350 (70 µm)]
  • Centrifuge that can spin 50‐ml and 15‐ml tubes
  • MACS preseparation filters (Miltenyi Biotec, cat. no. 130‐041‐407)
  • 37°C humidified incubator with 5% CO 2
  • Inverted phase‐contrast microscope
NOTE: It is important to test different lots of collagenase, as there is lot‐to‐lot variation in the specific activity of collagenase, and contaminating enzymes, such as trypsin and clostripain. Worthington Biochemical has a collagenase sampling program to determine which lot is most appropriate for your needs, and then permits selection of specific lots based on enzyme activity.

Alternate Protocol 1: Two‐Step Digestion Method to Increase the Proportion of Myogenic Cells

  • Collagenase, Type 4 (Worthington Biochemical)
NOTE: It is important to test different lots of collagenase, as there is lot‐to‐lot variation in the specific activity of collagenase, and contaminating enzymes, such as trypsin and clostripain. Worthington Biochemical has a collagenase sampling program to determine which lot is most appropriate for your needs, and then permits selection of specific lots based on enzyme activity. For the first digestion, you will want a lot with low clostripain activity, to prevent removal of the basal lamina from the fibers. For the second digestion, you will use the lot from the one‐step digestion method.

Support Protocol 1: Cell Counting Using a Hemacytometer and Setting up Dilutions for Culture

  Materials
  • Cell suspension (see protocol 1)
  • Hemacytometer coverslips
  • Hemacytometer
  • 20‐µl pipettor and appropriate tips
  • Inverted microscope to count cells on hemacytometer

Support Protocol 2: Generating Delta‐1ext‐IgG and Human IgG‐Coated Plates

  Materials
  • Delta‐1ext‐IgG: prepared as a 1.2 mg/ml solution and frozen in 500‐µl aliquots at −80°C
  • Human IgG: prepared as a 1 mg/ml solution and frozen in 500‐µl aliquots at −80°C (Sigma‐Aldrich, cat. no. I‐4506)
  • Ice
  • Dulbecco's phosphate‐buffered saline (D‐PBS; calcium‐ and magnesium‐free)
  • Growth medium (see recipe) or 2% bovine serum albumin (BSA) in D‐PBS (Sigma Aldrich, cat. no. A‐3294)
  • 10‐cm tissue culture‐treated polystyrene plates
  • Parafilm
  • 4°C refrigerator or cold room
  • 37°C humidified incubator with 5% CO 2

Support Protocol 3: Determining the Percentage of Myogenic Cells in the Freshly Isolated Cell Suspension by Counting Colonies

  Materials
  • Cells from protocol 1 or the Alternate Protocol
  • Growth medium (see recipe)
  • 10‐cm tissue culture treated polystyrene plates
  • Inverted phase‐contrast microscope
  • Object marker for microscope objective or Sharpie marker

Basic Protocol 2: Transplantation of Expanded Cells

  Materials
  • Plates of cells (see protocol 1 or the Alternate Protocol)
  • Dulbecco's phosphate‐buffered saline (D‐PBS; calcium‐ and magnesium‐free)
  • Cell dissociation buffer (Invitrogen, cat. no. 13151‐014)
  • 70% ethanol in a spray bottle
  • Liquid nitrogen
  • CO 2
  • OCT (Sakura Finetek, cat. no. 4583)
  • Dry ice
  • 37°C humidified incubator with 5% CO 2
  • Inverted phase‐contrast microscope
  • 15‐ml conical polystyrene tubes
  • 5‐ml pipets
  • Pipet‐aid
  • Centrifuge that can spin 50‐ml and 15‐ml tubes
  • 200‐µl pipettor and appropriate tips
  • Sterile microcentrifuge tubes
  • 0.5‐ml insulin syringe, or similar small volume syringe with 1/2‐in. needle
  • Aluminum block
  • Styrofoam boxes
  • Cryomolds (Sakura Finetek, cat. no. 4565)
  • Long forceps
  • Aluminum foil, cut into 10 × 10–cm pieces
  • Additional reagents and equipment for counting the cells (see protocol 3) and preparing the mice for transplantation (see protocol 7)

Support Protocol 4: Preparing Mice for Transplantation

  Materials
  • NOD/SCID mice (or other appropriate strain)
  • Injectable anesthesia (e.g., avertin, ketamine/xylazine)
  • 70% ethanol in a spray bottle
  • Holder for mice—to keep mice in one position and only expose hindlimbs to irradiation
  • Cesium‐137 source for irradiation
  • 0.5‐ml insulin syringe, or similar small volume syringe with 1/2‐in. needle
  • 1.2% barium chloride (Sigma‐Aldrich, cat. no. B0750)

Support Protocol 5: Analyzing Injected Muscle by Immunostaining of Cryosections

  Materials
  • Frozen muscles (see protocol 6)
  • Acetone, precooled to −20°C
  • Dulbecco's phosphate‐buffered saline (D‐PBS; calcium‐ and magnesium‐free)
  • Blocking buffer (see recipe)
  • 1° antibody dilution buffer (see recipe)
  • Primary antibodies including:
    • Lamin A/C antibody (Vector Laboratories, cat. no. VP‐L550)
    • Canine‐specific dystrophin antibody (MANDYS102 or MANDYS107; Developmental Studies Hybridoma Bank)
    • Pan dystrophin antibody (MANEX1A; Developmental Studies Hybridoma Bank)
  • Secondary antibody: e.g., AlexaFluor488‐labeled goat anti‐mouse IgG (Invitrogen, cat. no. A11001)
  • ProLong Gold Antifade with DAPI (Invitrogen, cat. no. P‐36931)
  • Cryostat
  • Superfrost Plus slides (Fisher Scientific, cat. no. 12‐550‐15)
  • Glass staining jars (e.g., Coplin jars)
  • Staining chamber
  • Hydrophobic marker (e.g., ImmEdge Pen; Vector Laboratories, cat. no. H‐4000)
  • Paper towels
  • Glass coverslips
  • Fluorescence microscope
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Figures

Videos

Literature Cited

Literature Cited
   Bischoff, R. 1986. Proliferation of muscle satellite cells on intact myofibers in culture. Dev. Biol. 115:129‐139.
   Carlson, B.M. 1968. Regeneration of the completely excised gastrocnemius muscle in the frog and rat from minced muscle fragments. J. Morphol. 125:447‐472.
   Collins, C.A., Olsen, I., Zammit, P.S., Heslop, L., Petrie, A., Partridge, T.A., and Morgan, J.E. 2005. Stem cell function, self‐renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122:289‐301.
   Conboy, I.M. and Rando, T.A. 2002. The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev. Cell 3:397‐409.
   Delaney, C., Heimfeld, S., Brashem‐Stein, C., Voorhies, H., Manger, R.L., and Bernstein, I.D. 2010. Notch‐mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat. Med. 16:232‐236.
   Gross, J.G., Bou‐Gharios, G., and Morgan, J.E. 1999. Potentiation of myoblast transplantation by host muscle irradiation is dependent on the rate of radiation delivery. Cell Tissue Res. 298:371‐375.
   Konigsberg, I.R. 1960. The differentiation of cross‐striated myofibrils in short term cell culture. Exp. Cell Res. 21:414‐420.
   Konigsberg, I.R. 1979. Skeletal myoblasts in culture. Methods Enzymol. 58:511‐527.
   Konigsberg, I.R., McElvain, N., Tootle, M., and Herrmann, H. 1960. The dissociability of deoxyribonucleic acid synthesis from the development of multinuclearity of muscle cells in culture. J. Biophys. Biochem. Cytol. 8:333‐343.
   Morgan, J.E., Hoffman, E.P., and Partridge, T.A. 1990. Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse. J. Cell Biol. 111:2437‐2449.
   Parker, M.H., Loretz, C., Tyler, A.E., Duddy, W.J., Hall, J.K., Olwin, B.B., Bernstein, I.D., Storb, R., and Tapscott, S.J. 2012a. Activation of notch signaling during ex vivo expansion maintains donor muscle cell engraftment. Stem Cells 30:2212‐2220.
   Parker, M.H., Loretz, C., Tyler, A.E., Snider, L., Storb, R., and Tapscott, S.J. 2012b. Inhibition of CD26/DPP‐IV enhances donor muscle cell engraftment and stimulates sustained donor cell proliferation. Skelet. Muscle 2:4.
   Partridge, T.A. and Sloper, J.C. 1977. A host contribution to the regeneration of muscle grafts. J. Neurol. Sci. 33:425‐435.
   Partridge, T.A., Grounds, M., and Sloper, J.C. 1978. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature 273:306‐308.
   Partridge, T.A., Morgan, J.E., Coulton, G.R., Hoffman, E.P., and Kunkel, L.M. 1989. Conversion of mdx myofibres from dystrophin‐negative to ‐positive by injection of normal myoblasts. Nature 337:176‐179.
   Qu, Z., Balkir, L., van Deutekom, J.C., Robbins, P.D., Pruchnic, R., and Huard, J. 1998. Development of approaches to improve cell survival in myoblast transfer therapy. J. Cell Biol. 142:1257‐1267.
   Rinaldini, L.M. 1959. An improved method for the isolation and quantitative cultivation of embryonic cells. Exp. Cell Res. 16:477‐505.
   Varnum‐Finney, B., Wu, L., Yu, M., Brashem‐Stein, C., Staats, S., Flowers, D., Griffin, J.D., and Bernstein, I.D. 2000. Immobilization of Notch ligand, Delta‐1, is required for induction of notch signaling. J. Cell Sci. 113 Pt 23:4313‐4318.
   Varnum‐Finney, B., Brashem‐Stein, C., and Bernstein, I.D. 2003. Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors with lymphoid and myeloid reconstituting ability. Blood 101:1784‐1789.
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