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

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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 (ALDH^hi ) 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 ALDH^hi 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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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 ALDH^hi 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 ALDH^lo and ALDH^hi human bone marrow cells ( protocol 1 ) and counting viable cells by trypan blue exclusion (unit 1.3 )

NOTE : For the expansion of HCFC, ALDH^hi 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 40 ¹³⁷ Cs 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 ALDH^lo and ALDH^hi 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 (O₂ )
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
ALDH^lo cells or ALDH^hi 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 (CuSO₄ )
- 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

Figure 2.B0.1 Schematic representation for the isolation and characterization of bone marrow–derived ALDH^hi mixed progenitor cells. Human bone marrow mononuclear cells (MNC) are isolated by Ficoll‐Hypaque centrifugation, and incubated with Aldefluor reagent. Using fluorescence activated cell sorting, MNC are purified into ALDH^lo cell and ALDH^hi cell populations based on relative ALDH activity. Cell‐surface phenotype is analyzed by flow cytometry, and clonal colony‐forming unit assays are performed to calculate relative frequency of hematopoietic, endothelial, or mesenchymal progenitor cells. ALDH^hi progenitor cells can be expanded efficiently in lineage‐specific culture. ALDH^hi mixed progenitor cells or their expanded progeny can then be transplanted into the tail vein or into the pancreas of hyperglycemic NOD/SCID mice to assess their capacity to stimulate endogenous islet regeneration.

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Figure 2.B0.2 In vivo model of human progenitor cell–stimulated beta cell regeneration after transplantation into hyperglycemic mice. NOD/SCID mice are intraperitoneally injected with 35 mg/kg streptozotocin (STZ) from days 1 to 5 to induce beta‐cell deletion and subsequent hyperglycemia. On day 10, mice with systemic blood glucose levels between 15 and 25 mmol/liter are sublethally irradiated (300 cGy) and transplanted with human bone marrow–derived progenitor cells. Blood glucose concentrations are monitored weekly for 42 days. At a time point 24 hr prior to euthanasia, mice are intraperitoneally injected with 200 µg EdU to label dividing cells, and a fasted glucose tolerance test is performed. On day 42, murine blood is collected by cardiac puncture and serum is separated by centrifugation and cryopreserved for serum insulin quantification by ELISA. Following euthanasia, bone marrow is collected for flow cytometry, while the spleen and pancreas are collected and segmented for the analysis of human cell engraftment by flow cytometry, or frozen in O.C.T. (Optimal Cutting Temperature) compound for subsequent sectioning and immunohistochemical analysis.

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Figure 2.B0.3 Intrapancreatic transplantation surgery. Representative photographs and step‐by‐step description of the intrapancreatic transplantation procedure.

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Figure 2.B0.4 Immunohistochemical analysis of islet regeneration. Human progenitor cell–transplanted NOD/SCID mice are euthanized, and the splenic portion of the pancreas frozen in OCT. Using a cryostat, the pancreas is sectioned into 10‐µm sections, with three sections (150 µm apart) per glass slide. Pancreatic tissue sections are analyzed for: islet size, islet number, and beta cell mass (staining for insulin); human cell engraftment (co‐staining for HLA‐A,B,C and insulin); islet vascularization (co‐staining for insulin and vWF or CD31); beta cell proliferation (co‐staining for insulin and EdU); and islet location (co‐staining for insulin and CK19).

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

Abstract

Table of Contents

Materials

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

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Literature Cited