Transplantation Models to Characterize the Mechanisms of Stem Cell–Induced Islet Regeneration

Gillian I. Bell1, Ayesh K. Seneviratne1, Grace N. Nasri1, David A. Hess1

1 The University of Western Ontario, London, Ontario
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.4
DOI:  10.1002/9780470151808.sc02b04s26
Online Posting Date:  September, 2013
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Abstract

ABSTRACT

This unit describes our current knowledge regarding the isolation human bone marrow–derived progenitor cells for the paracrine stimulation of islet regeneration after transplantation into immunodeficient mouse models of diabetes. By using high aldehyde dehydrogenase (ALDHhi) activity, a conserved function in multiple stem cell lineages, a mixed population of hematopoietic, endothelial, and mesenchymal progenitor cells can be efficiently purified using flow cytometry. We describe in vitro approaches to characterize and expand these distinct cell types. Importantly, these cell types can be transplanted into immunodeficient mice rendered beta‐cell deficient by streptozotocin (STZ) treatment, in order monitor functional recovery from hyperglycemia and to characterize endogenous islet regeneration via paracrine mechanisms. Herein, we provide detailed protocols for: (1) isolation and characterization of ALDHhi cells for the establishment of hematopoietic and multipotent‐stromal progenitor lineages; (2) intravenous and intrapancreatic transplantation of human stem cell subtypes for the quantification of glycemic recovery in STZ‐treated immunodeficient mice; and (3) immunohistochemical characterization of islet recovery via the stimulation of islet neogenic, beta‐cell proliferative, and islet revascularization programs. Collectively, these systems can be used to support the pre‐clinical development of human progenitor cell–based therapies to treat diabetes via islet regeneration. Curr. Protoc. Stem Cell Biol. 26:2B.4.1‐2B.4.35. © 2013 by John Wiley & Sons, Inc.

Keywords: aldehyde dehydrogenase; stem cells; bone marrow; transplantation; hematopoietic progenitor cells; multipotent stromal cells; islet regeneration; diabetes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Characterization of Bone Marrow Progenitor Cells with High Aldehyde Dehydrogenase Activity
  • Support Protocol 1: Phenotypic Analysis of ALDHhi Progenitor Cells
  • Support Protocol 2: Colony‐Forming Cell Assays and Expansion
  • Basic Protocol 2: Transplantation of ALDHhi Cells into Hyperglycemic Mice
  • Alternate Protocol 1: Intrapancreatic Transplantation of ALDHhi or ALDHlo Progenitor Cells
  • Support Protocol 3: Mouse Euthanasia and Tissue Dissection
  • Support Protocol 4: Serum Insulin Quantification Using ELISA
  • Support Protocol 5: Analysis of Human Cell Engraftment by Flow Cytometry
  • Basic Protocol 3: Immunohistochemical Analysis of Islet Regeneration
  • Support Protocol 6: Quantification of Islet Size, Islet Number, and Beta Cell Mass
  • Support Protocol 7: Analyses of Human Cell Association with Islets
  • Support Protocol 8: Analyses of Islet Blood Vessel Density
  • Support Protocol 9: Analyses of Islet Cell Proliferation
  • Support Protocol 10: Analysis of Islet Association with CK19+ Ducts
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation and Characterization of Bone Marrow Progenitor Cells with High Aldehyde Dehydrogenase Activity

  Materials
  • Human bone marrow (BM) donors or BM from commercial source (Lonza)
  • Dulbecco's phosphate buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250), warmed to room temperature
  • Ficoll‐Hypaque gradient (density 1.077 g/liter; Fisher Scientific, cat. no.17144003)
  • Beckman GPR centrifuge with horizontal rotor
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250) containing 5% fetal bovine serum (FBS)
  • Ammonium chloride solution (Stem Cell Technologies, cat. no. 20110)
  • Aldefluor Kit (Stem Cell Technologies, cat. no. 01700) including:
    • Aldefluor assay buffer
    • Dry Aldefluor reagent, 50 µg
    • 2 N HCl
    • DMSO
    • DEAB reagent, 1.5 mM, in 95% ethanol
  • 15‐ and 50‐ml conical tubes (e.g., BD Falcon)
  • Beckman GPR centrifuge with horizontal rotor
  • 50‐µm PARTEC CellTrics filters (http://www.partec.com)
  • High‐speed fluorescence activated cell sorter (FACS Aria III, Becton Dickinson; also see Coligan et al., , Chapter 5)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (unit 1.3) and flow cytometry (Coligan et al., , Chapter 5)

Support Protocol 1: Phenotypic Analysis of ALDHhi Progenitor Cells

  Materials
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250) containing 5% (v/v) fetal bovine serum (FBS)
  • Desired fluorescently labeled anti‐human antibodies and isotype controls (Table 2.4.1)
  • 7‐AAD Staining Solution (BD Biosciences, cat. no. 559925)
  • 5‐ml polystyrene plastic tubes (e.g., BD Falcon)
  • Flow cytometer: FACSCalibur (Becton Dickinson)
  • FlowJo analysis software (http://flowjo.com/)
  • Additional reagents and equipment for flow cytometry (Coligan et al., , Chapter 5) and isolation of ALDHhi cells ( protocol 1)
Table 2.0.1   MaterialsAntibody Clones, Fluorochromes, and Amounts Used to Analyze Hematopoietic Progenitor Cell Surface Marker Expression

Cocktail name Antibodies (clone, antibody, fluorochrome) Suggested concentration/tube (ng/ml)
Isotype MOPC‐21, IgG1, APC 44
MOPC‐21, IgG1, FITC 250
MOPC‐21, IgG1, PE 25
7AAD 250
Progenitor 1 HI30, CD45, APC 44
581, CD34, FITC 250
H1T2, CD38, PE 25
7AAD 250
Progenitor 2 HI30, CD45, APC 44
2B8, CD117, FITC 250
CD133, CD133, PE 25
7AAD 250
Myeloid/monocyte HI30, CD45, APC 44
M5E2, CD14, FITC 250
WM53, CD33, PE 25
7AAD 250

Support Protocol 2: Colony‐Forming Cell Assays and Expansion

  Materials
  • H4434 Methocult medium (StemCell Technologies, cat. no. 04434)
  • Iscove's modified Dulbecco's medium (IMDM; Invitrogen, cat. no. 12440‐046)
  • HCFC expansion medium: X‐Vivo 15 medium (Lonza, cat. no. 04‐744Q) supplemented with 10 ng/ml Flt‐3 ligand, SCF, and TPO
  • EGM2‐Bulletkit (to prepare ECFC expansion medium; Lonza, cat. no. CC‐3162) including:
    • EBM‐2 basal medium, 500 ml
    • Fetal bovine serum (FBS), 10 ml
    • Hydrocortisone, 0.2 ml
    • hFGF‐B, 2 ml
    • VEGF, 0.5 ml
    • IGF‐1, 0.5 ml
    • hEGF, 0.5 ml
    • Heparin, 0.5 ml
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250)
  • AmnioMax complete medium (MCFC expansion medium):
    • AmnioMAX C‐100 basal medium (Invitrogen, cat. no. 17001074)
    • AmnioMAX C‐100 supplement (Invitrogen, cat. no. 12556023)
  • TrypLE Express (Life Technologies)
  • 1‐ml syringes with16‐G needles
  • 12‐well suspension plates (BD Falcon, cat. no. 351143)
  • Hand tally counter (multi‐channel) for scoring colonies
  • Collagen I–coated 6‐well plates (BD Biosciences, cat no. 356400)
  • Inverted light microscope
  • 25‐ and 75‐cm2 tissue culture flasks (Corning)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Additional reagents and equipment for obtaining ALDHlo and ALDHhi human bone marrow cells ( protocol 1) and counting viable cells by trypan blue exclusion (unit 1.3)
NOTE: For the expansion of HCFC, ALDHhi cells are plated in serum‐free liquid medium (X‐Vivo 15 medium plus 10 ng/ml SCF, Flt3L, and TPO) rather than methylcellulose medium. For expansion of ECFC and MCFC use EGM‐2 Bulletkit and AmnioMax complete medium, respectively.

Basic Protocol 2: Transplantation of ALDHhi Cells into Hyperglycemic Mice

  Materials
  • Citric acid, anhydrous (Sigma, cat. no.C2404‐100G)
  • Autoclaved filter‐sterilized double‐distilled water
  • Trisodium citrate dihydrate (Sigma, cat. no. S1804)
  • 10 mM NaOH
  • NOD/SCID mice (The Jackson Laboratory)
  • Streptozotocin (STZ) (Sigma‐Aldrich, cat. no. S0130)
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+, no Mg2+; Life Technologies, cat. no. 14190250)
  • TrypLE Express (Invitrogen, cat. no. 12604013)
  • MSC growing in culture (expanded as described in protocol 3)
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250) containing 5% (v/v) fetal bovine serum (FBS)
  • 10 mM 5‐ethynyl‐2″‐deoxyuridine (EdU; Invitrogen, cat. no. A10044) in DPBS without Ca or Mg (store up to 1 year at –20°C)
  • 0.25 mg/ml dextrose, anhydrous (VWR, cat. no. CABDH0230‐500G): add 250 ml filter‐sterilized double‐distilled water to 62.5 g dextrose; mix until dextrose is dissolved; sterilize this solution with a 0.2‐µm syringe filter and store at room temperature
  • 600‐ml Pyrex glass beaker
  • Magnetic stirrer and stir bar
  • 0.2‐µm sterile syringe filters
  • Animal balance
  • Insulin syringes with 28‐G needles (Fisher Scientific, cat. no. 14‐829‐1B)
  • FreeStyle blood glucose test strips and glucose monitor (Abbott Diabetes Inc., http://www.abbottdiabetescare.com/)
  • Inverted light microscope
  • Gammacell 40 137Cs irradiator (MDS Nordion, http://www.nordion.com)
  • Syringes with 30‐G needles for tail venipuncture
  • Beckman GPR centrifuge with horizontal rotor
  • Additional reagents and equipment for injection of mice (Donovan and Brown, ), obtaining ALDHlo and ALDHhi human bone marrow cells ( protocol 1), and counting viable cells by trypan blue exclusion (unit 1.3)

Alternate Protocol 1: Intrapancreatic Transplantation of ALDHhi or ALDHlo Progenitor Cells

  Materials
  • Isoflurane
  • Oxygen (O 2)
  • Eye lubricant (Allergan Refresh Lacrilube, http://www.allergan.com/)
  • Buprenorphine: purchased from the Western University animal care and veterinary services facility as a stock solution of 0.3 mg/ml; the stock is then diluted in normal saline to a 0.012 mg/ml working solution (both the stock and the working solution are stored at room temperature)
  • Ketoprofen
  • Hair removal cream (Nair)
  • Bacteriostatic soap
  • Isopropyl alcohol
  • Betadine solution
  • ALDHlo cells or ALDHhi cells ( protocol 1)
  • Dulbecco's phosphate buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250)
  • Vetbond tissue adhesive
  • Mobile laboratory animal anesthesia system (VetEquip, cat. no. 901807), including nose cones
  • Electric hair clippers
  • Gauze
  • Sterile cotton tipped applicators
  • Surgical scissors and forceps
  • Microinjection syringes (Becton Dickinson Ultra Fine II, 0.5 ml, 30‐G, cat. no. 320468)
  • 5‐0 silk sutures
NOTE: Perform 42‐day in vivo protocol as described in protocol 4. The following alternate protocol explains an adapted transplantation procedure on day 10 only.NOTE: For additional details and photographs of the intrapancreatic injection surgical procedures please refer to Figure .

Support Protocol 3: Mouse Euthanasia and Tissue Dissection

  Materials
  • Mice that have been subjected to EdU injection and glucose tolerance test ( protocol 4)
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250) containing 5% (v/v) fetal bovine serum (FBS)
  • Isoflurane, USP (Baxter)
  • Optimal cutting temperature (OCT) compound (Fisher Scientific, cat. no. 1437365)
  • Dry ice
  • 5‐ml polystyrene tubes
  • 15 mm × 15 mm tissue molds (Fisher Scientific, cat. no.22038217)
  • Gauze
  • Nalgene jar with cover (Thermo Scientific, cat. no. 5352‐0004)
  • Sterile 1‐ml syringe and 23‐G needle
  • Forceps
  • Dissection scissors
  • AB135‐S/FACT scale (Mettler Toledo)
  • IEC CL31 multispeed centrifuge Classic Plus (Thermo Scientific)
  • 40‐µm cell strainers
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Support Protocol 4: Serum Insulin Quantification Using ELISA

  Materials
  • Insulin (mouse) ultrasensitive EIA (ALPCO, cat. no. 80‐INSMSU‐E01, http://www.alpco.com/):
    • Insulin microplate
    • Zero standard (0 ng/ml)
    • Insulin standards (0.025, 0.063, 0.188, 0.5, 1.25, 3.75, 6.9 ng/ml)
    • Mammalian insulin high and low controls
    • Conjugate (11× HRP‐labeled monoclonal anti‐insulin antibody)
    • Conjugate buffer
    • Wash buffer (21×)
    • TMB substrate
    • Stop solution
    • Plate sealer
  • Serum samples ( protocol 6, step 8)
  • Plate shaker
  • Wash bottle
  • Microplate reader (Multiskan Ascent from Thermo Scientific)
  • Ascent software (Thermo Scientific)
NOTE: The following protocol has been modified from the manufacturer's instructions to utilize a wider range of standards.

Support Protocol 5: Analysis of Human Cell Engraftment by Flow Cytometry

  Materials
  • Mechanically triturated pancreas cells ( protocol 6, step 9)
  • Fluorescently labeled antibodies:
    • FITC‐labeled anti‐human CD45 antibody (4 µl/200 µl cells; BD Biosciences, cat. no. 555482)
    • PE‐labeled anti‐human HLA‐A,B,C antibody (4 µl/200 µl cells; BD Biosciences, cat. no. 555553)
    • Mouse IgG1 FITC antibody (4 µl/200 µl cells; BD Biosciences, cat. no. 555748)
    • Mouse IgG1 PE antibody (4 µl/200 µl cells; BD Biosciences, cat. No. 555749)
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250) containing 5% fetal bovine serum (FBS)
  • 7‐AAD (5 µl/200 µl cells; BD Biosciences, cat. no. 559925)
  • 5‐ml polystyrene tubes
  • Centrifuge with horizontal rotor (Beckman)
  • BD FACSCalibur flow cytometer (BD Biosciences; also see Coligan et al., , Chapter 5)
  • FlowJo analysis software (http://flowjo.com/)
  • Additional reagents and equipment for flow cytometry ( protocol 2; also see Coligan et al., , Chapter 5)

Basic Protocol 3: Immunohistochemical Analysis of Islet Regeneration

  Materials
  • OCT‐embedded pancreatic tissue block ( protocol 6)
  • 10% formalin (Fisher Scientific, cat. no. SF98‐4)
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250)
  • 1% permeabilization buffer: DPBS (no Ca or Mg) containing 1% (v/v) Triton X‐100
  • SuperFrost Plus glass slides (Menzel‐Gläser; http://www.menzel.de/)
  • Leica CM1850 cryostat
  • Microtome blades
  • Vacuum flask
  • Fine‐tip black marker

Support Protocol 6: Quantification of Islet Size, Islet Number, and Beta Cell Mass

  Materials
  • Fixed and permeabilized pancreas tissue slides ( protocol 9)
  • Peroxidase blocking solution: DPBS (no Ca or Mg; Life Technologies, cat. no. 14190250) containing 0.3% hydrogen peroxide (final) and 0.1% FBS
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250)
  • Mouse on mouse (M.O.M.) basic kit (Vector Labs, cat. no. BMK‐2202) including:
    • M.O.M. mouse IgG blocking reagent
    • M.O.M. diluent
    • M.O.M. protein concentrate
  • Monoclonal anti‐insulin antibody (Sigma, cat. no. I2018)
  • Horse anti‐mouse IgG antibody, peroxidase‐labeled (Vector Labs, cat. no. PI‐2007)
  • ImmPACT DAB peroxidase substrate (Vector Labs, cat. no. SK‐4105)
  • Hematoxylin QS (Vector Labs, cat. no. H‐3404)
  • VectaMount mounting medium (Vector Labs, cat. no. H5000)
  • Slide rack
  • Plate shaker
  • Glass coverslips
  • AxioScope Z2 light microscope (Zeiss)
  • Northern Eclipse software (EMPIX, http://www.empix.com/)
  • AxioVision 4.7.2 Software (Zeiss)

Support Protocol 7: Analyses of Human Cell Association with Islets

  Materials
  • Fixed and permeabilized pancreas tissue slides ( protocol 9)
  • Mouse on mouse (M.O.M.) basic kit (Vector Labs, cat. no. BMK‐2202) including:
    • M.O.M. mouse IgG blocking reagent
    • M.O.M. diluent
    • M.O.M. protein concentrate
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250)
  • Mouse anti‐human HLA‐A,B,C antibody (BD Biosciences, cat. no. 555551)
  • Horse anti‐mouse IgG fluorescein antibody (Vector Labs, cat. no. FI‐2000)
  • Goat blocking buffer: 2.5 ml goat serum (Vector Labs, cat. no. S 1000) in 47.5 ml DPBS without Ca or Mg), prepared in advance
  • Rabbit anti‐mouse insulin antibody (Santa Cruz Biotechnology, cat. no. sc‐9168)
  • Goat anti‐rabbit Texas Red IgG antibody (Vector Labs, cat. no. TI‐1000)
  • Vectashield mounting medium with DAPI (Vector Labs, cat. no. H‐1200)
  • Clear nail polish
  • Slide rack
  • Plate shaker
  • Glass coverslips
  • Slide box
  • AxioScope Z2 light microscope (Zeiss)
  • AxioVision 4.7.2 Software (Zeiss)

Support Protocol 8: Analyses of Islet Blood Vessel Density

  Materials
  • Fixed and permeabilized pancreas tissue slides ( protocol 9)
  • Mouse on mouse (M.O.M.) basic kit (Vector Labs, cat. no. BMK‐2202) including:
    • M.O.M. mouse IgG blocking reagent
    • M.O.M. diluent
    • M.O.M. protein concentrate
  • Monoclonal anti‐insulin antibody (Sigma, cat. no. I2018)
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250)
  • Horse anti‐mouse IgG Texas Red antibody (Vector Labs, cat. no. TI‐2000)
  • Goat blocking buffer: 2.5 ml goat serum (Vector Labs, cat. no. S 1000) in 47.5 ml DPBS without Ca or Mg or rabbit blocking buffer: 2.5 ml rabbit serum (Vector Labs, cat. no. S 5000) in 47.5 ml DPBS without Ca or Mg), prepared in advance
  • Rabbit anti‐mouse vWF antibody (Santa Cruz Biotechnology, cat. no. sc‐14014) or rat anti‐mouse CD31 antibody (BD Biosciences, cat no. 550274)
  • Goat anti‐rabbit IgG fluorescein antibody (Vector Labs, cat. no. FI‐1000) or rabbit anti‐rat IgG fluorescein antibody (Vector Labs, cat. no. FI‐4000)
  • Vectashield mounting medium with DAPI (Vector Labs, cat. no. H‐1200)
  • Clear nail polish
  • Slide rack
  • Slide box
  • AxioScope Z2 light microscope (Zeiss)
  • AxioVision 4.7.2 Software (Zeiss)

Support Protocol 9: Analyses of Islet Cell Proliferation

  Materials
  • Click‐iT EdU Imaging Kit (Life Technologies, cat. no. C10337) including:
    • 5‐ethynyl‐2″‐deoxyuridine (EdU)
    • Alexa Fluor 488 azide
    • Dimethylsulfoxide (DMSO)
    • Click‐iT EdU reaction buffer
    • Copper sulfate (CuSO 4)
    • Click‐iT EdU buffer additive
  • Fixed and permeabilized pancreas tissue slides ( protocol 9)
  • Vectashield mounting medium with DAPI (Vector Labs, cat. no. H‐1200)
  • Clear nail polish
  • Slide rack
  • Glass coverslips
  • AxioScope Z2 light microscope (Zeiss)
  • AxioVision 4.7.2 Software (Zeiss)

Support Protocol 10: Analysis of Islet Association with CK19+ Ducts

  Materials
  • Fixed and permeabilized pancreas tissue slides ( protocol 9)
  • Mouse on mouse (M.O.M.) basic kit (Vector Labs, cat. no. BMK‐2202) including:
    • M.O.M. mouse IgG blocking reagent
    • M.O.M. diluent
    • M.O.M. protein concentrate
  • Monoclonal anti‐insulin antibody (Sigma, cat. no. I2018)
  • Dulbecco's phosphate‐buffered saline (DPBS, no Ca2+ no Mg2+; Life Technologies, cat. no. 14190250)
  • Horse anti‐mouse IgG Texas Red antibody (Vector Labs, cat. no. TI‐2000)
  • Goat blocking buffer: 2.5 ml goat serum (Vector Labs, cat. no. S 1000) in 47.5 ml DPBS without Ca or Mg
  • Rabbit anti‐mouse CK19 antibody (Abcam, cat. no. ab‐15463)
  • Goat anti‐rabbit IgG fluorescein antibody (Vector Labs, cat. no. FI‐1000)
  • Vectashield mounting medium with DAPI (Vector Labs, cat. no. H‐1200)
  • Clear nail polish
  • Slide rack
  • Glass coverslips
  • AxioScope Z2 light microscope (Zeiss)
  • AxioVision 4.7.2 Software (Zeiss)
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Figures

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Literature Cited

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Key References
  Bell et al., 2012a. See above.
  This paper demonstrates that sequential transplantation of MSC followed by ALDHhi cells improved hyperglycemia and glucose tolerance by increasing beta cell mass via stimulation of islet‐regenerative and revascularization program
  Bell et al., 2012b. See above.
  This paper demonstrates that endogenous islet recovery after progenitor cell transplantation can occur via islet proliferative and neogenic mechanisms modulated by subtypes of progenitor cells administered.
  Bell et al., 2012c. See above.
  This paper demonstrates that intra‐pancreatic delivery of umbilical cord blood‐derived ALDHhi cells potentiated islet‐associated cell proliferation and revascularization, resulting in the recovery of host islet function.
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