Isolation and Characterization of Mesoangioblasts from Mouse, Dog, and Human Tissues

Rossana Tonlorenzi1, Arianna Dellavalle1, Esther Schnapp1, Giulio Cossu1, Maurilio Sampaolesi1

1 Stem Cell Research Institute, San Raffaele Scientific Institute, Milan, Italy
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.1
DOI:  10.1002/9780470151808.sc02b01s3
Online Posting Date:  December, 2007
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Abstract

Mesoangioblasts are recently identified stem/progenitor cells, associated with small vessels of the mesoderm in mammals. Originally described in the mouse embryonic dorsal aorta, similar though not identical cells have been later identified and characterized from postnatal small vessels of skeletal muscle and heart (not described in this unit). They have in common the anatomical location, the expression of endothelial and/or pericyte markers, the ability to proliferate in culture, and the ability to undergo differentiation into various types of mesoderm cells upon proper culture conditions. Currently, the developmental origin of mesoangioblasts, their phenotypic heterogeneity, and the relationship with other mesoderm stem cells are not understood in detail and are the subject of active research. However, from a practical point of view, these cells have been successfully used in cell transplantation protocols that have yielded a significant rescue of structure and function in skeletal muscle of dystrophic mice and dogs. Since the corresponding human cells have been recently isolated and characterized, a clinical trial with these cells is planned in the near future. This unit provides detailed methods for isolation, culture, and characterization of mesoangioblasts. Curr. Protoc. Stem Cell Biol. 3:2B.1.1‐2B.1.29. © 2007 by John Wiley & Sons, Inc.

Keywords: mesoangioblasts; pericytes; mesoderm progenitor cells; cell culture

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation, Cloning, and Propagation of Mesoangioblasts from Mouse Embryonic Aorta
  • Basic Protocol 2: Isolating, Cloning, Propagating, and Freezing of Mouse Adult Muscle Mesoangioblasts
  • Alternate Protocol 1: Isolation, Propagation, and Cloning of Human Adult Pericyte‐Derived Cells
  • Alternate Protocol 2: Isolation and Cloning of Adult Dog Skeletal Muscle Mesoangioblasts
  • Basic Protocol 3: Differentiation of Mesoangioblasts: Co‐Culture with Murine C2C12 Myoblasts
  • Alternate Protocol 3: Differentiation of Canine Mesoangioblasts or Human Pericyte‐Derived Cells: Co‐Culture with L6 Rat Myoblasts
  • Alternate Protocol 4: Differentiation of Human Pericyte‐Derived Cells: Spontaneous Skeletal Myogenic Differentiation
  • Alternate Protocol 5: Differentiation of Mesoangioblast/Pericyte‐Derived Cells: Induction of Smooth Muscle Cell Differentiation by TGFβ Treatment
  • Alternate Protocol 6: Differentiation of Mesoangioblast/Pericyte‐Derived Cells: Induction of Osteoblast Differentiation by BMP2 Treatment
  • Alternate Protocol 7: Differentiation of Mesoangioblast/Pericyte‐Derived Cells: Induction of Adipocyte Differentiation
  • Support Protocol 1: Preparation of Mitotically Inactive STO Feeder Layer
  • Support Protocol 2: Collagen Coating of Tissue Culture Surfaces
  • Support Protocol 3: Matrigel Coating of Tissue Culture Surfaces
  • Support Protocol 4: Freezing Mesoangioblasts and Human Pericyte‐Derived Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation, Cloning, and Propagation of Mesoangioblasts from Mouse Embryonic Aorta

  Materials
  • Dissected aorta (three mouse embryos at embryonic day 10.5)
  • D20 medium (see recipe), sterile
  • 3.5‐cm collagen‐coated petri dishes (see protocol 12)
  • Phosphate‐buffered saline without Ca2+/Mg2+ (CMF‐PBS; Sigma cat. no. D8537), sterile
  • Collagenase/dispase solution (see recipe, Sigma), sterile
  • Fetal bovine serum (heat‐inactivated FBS; Cambrex), sterile
  • Trypan blue (Sigma cat. no. T8154)
  • 48‐well plates (Nunc) coated with mitotically inactivated STO cells (see protocol 11)
  • 0.05%/0.02% (w/v) trypsin/EDTA (Sigma cat. no. T3924), sterile
  • Curved and straight forceps, sterile
  • Rounded‐edge disposable scalpels, sterile
  • 3.5‐, 6‐, and 15‐cm petri dishes
  • 1‐ml sterile syringes and insulin needles
  • 37°C, 5% CO 2/5% O 2/90% N 2 humidified (water‐saturated) incubator
  • 15‐ml centrifuge tubes
  • 37°C water bath
  • Hemacytometer
  • Dissection microscope
  • 48‐well plates
  • 25‐ and 75‐cm2 vented tissue culture flasks (Nunc)
  • Additional reagents and equipment for trypan blue exclusion (unit 1.3)

Basic Protocol 2: Isolating, Cloning, Propagating, and Freezing of Mouse Adult Muscle Mesoangioblasts

  Materials
  • Mouse skeletal (Tibialis anterior) muscle fragments (≥30 mg)
  • Phosphate‐buffered saline without Ca2+/Mg2+, (CMF‐PBS; Sigma), sterile
  • M5 medium (see recipe), sterile
  • D20 medium (see recipe), sterile
  • Collagenase/dispase solution (see recipe), sterile
  • Trypan blue (Sigma)
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA, (Sigma), sterile
  • 6‐, 10‐, and 15‐cm petri dishes (Nunc)
  • Rounded‐edge disposable scalpels, sterile
  • Curved forceps, sterile
  • 5% CO 2, 5% O 2, 90% N 2 incubator
  • Additional reagents and equipment for tissue processing (see protocol 1)

Alternate Protocol 1: Isolation, Propagation, and Cloning of Human Adult Pericyte‐Derived Cells

  • Skeletal muscle fragments (≥100 mg) from a muscle biopsy
  • M5 medium (see recipe), sterile

Alternate Protocol 2: Isolation and Cloning of Adult Dog Skeletal Muscle Mesoangioblasts

  Materials
  • C2C12 cells grown in a 25‐cm2 tissue culture flask (ATCC #CRL‐1772)
  • Phosphate‐buffered saline without Ca2+/Mg2+ (CMF‐PBS; Sigma), sterile
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA (Sigma), sterile
  • D20 medium (see recipe)
  • D10 medium (see recipe)
  • Mesoangioblasts to be tested
  • D2 medium (see recipe)
  • 4% (w/v) paraformaldehyde (PFA)
  • 37°C, 5% CO 2 incubator
  • 3.5‐cm petri dishes
  • Additional reagents and equipment for trypan blue exclusion (unit 1.3)

Basic Protocol 3: Differentiation of Mesoangioblasts: Co‐Culture with Murine C2C12 Myoblasts

  • L6 cells grown in a 25‐cm2 tissue culture flask
  • Mesoangioblasts to be tested grown in a 25‐cm2 tissue culture flask
  • M5 medium (see recipe)
  • 15‐ml centrifuge tubes

Alternate Protocol 3: Differentiation of Canine Mesoangioblasts or Human Pericyte‐Derived Cells: Co‐Culture with L6 Rat Myoblasts

  • Dog mesoangioblasts/human pericyte‐derived cells to be tested grown in a 25‐cm2 tissue culture flask
  • M5 medium (see recipe)
  • Reduced growth factor Matrigel–coated 3.5‐cm petri dishes (see protocol 13), freshly prepared

Alternate Protocol 4: Differentiation of Human Pericyte‐Derived Cells: Spontaneous Skeletal Myogenic Differentiation

  • Mesoangioblasts/human pericyte‐derived cells to be tested grown in a 25‐cm2 tissue culture flask
  • M5 medium (see recipe)
  • 5 µg/ml TGFβ stock solution

Alternate Protocol 5: Differentiation of Mesoangioblast/Pericyte‐Derived Cells: Induction of Smooth Muscle Cell Differentiation by TGFβ Treatment

  • Mesoangioblast/human pericyte‐derived cell cultures to be tested grown in a 25‐cm2 tissue culture flask
  • 10 µg/ml BMP2 stock solution
  • Alkaline phosphatase staining solution (see recipe), freshly prepared

Alternate Protocol 6: Differentiation of Mesoangioblast/Pericyte‐Derived Cells: Induction of Osteoblast Differentiation by BMP2 Treatment

  • Mesoangioblast/human pericyte‐derived cell cultures to be tested grown in a 25‐cm2 tissue culture flask
  • Adypogenic induction medium (Cambrex)
  • Oil Red O solution (see recipe)
  • Inverted phase‐contrast microscope

Alternate Protocol 7: Differentiation of Mesoangioblast/Pericyte‐Derived Cells: Induction of Adipocyte Differentiation

  Materials
  • Frozen vials of STO (ATCC # CRL‐1503)
  • D10 medium (see recipe)
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA (Sigma), sterile
  • Mitomycin C stock solution (MMC, see recipe)
  • Phosphate‐buffered saline without Ca2+/Mg2+ (CMF‐PBS; Sigma), sterile
  • 15‐ml centrifuge tube
  • 75‐cm2 vented tissue culture flasks (Nunc)
  • 37°C, 5% CO 2 incubator
  • Additional reagents and equipment for trypan blue staining (unit 1.3)

Support Protocol 1: Preparation of Mitotically Inactive STO Feeder Layer

  Materials
  • Collagen type I solution (see recipe)
  • Petri dishes
  • 30°C oven

Support Protocol 2: Collagen Coating of Tissue Culture Surfaces

  Materials
  • Reduced growth factors Matrigel stock solution (BD Biosciences; see recipe)
  • High‐glucose DMEM (Sigma), ice cold
  • Petri dishes
  • 37°C, 5% CO 2 incubator

Support Protocol 3: Matrigel Coating of Tissue Culture Surfaces

  Materials
  • Murine and canine mesoangioblasts cultures grown in a 25‐cm2 tissue culture flask or human pericyte‐derived cell cultures grown in a 75‐cm2 tissue culture flask
  • D20 or M5 medium (see reciperecipes)
  • Freezing solution (see recipe), freshly prepared, ice cold
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA (Sigma), sterile
  • DMSO (Sigma), sterile
  • Hemacytometer
  • 1.8‐ml sterile cryovials (Corning), ice cold
  • Cryogenic‐controlled rate freezing container (Nalgene) or insulated cardboard/polystyrene foam box
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Figures

Videos

Literature Cited

Literature Cited
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