Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation into Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyses

Claudia Bruedigam1, Marjolein van Driel1, Marijke Koedam1, Jeroen van de Peppel1, Bram C.J. van der Eerden1, Marco Eijken1, Johannes P.T.M. van Leeuwen1

1 Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.3
DOI:  10.1002/9780470151808.sc01h03s17
Online Posting Date:  June, 2011
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Abstract

This unit describes basic techniques in human mesenchymal stem cell (hMSC) cultures. It includes protocols for the differentiation of hMSCs into osteogenic and adipogenic lineages, genetic perturbations, and phenotypic analyses. hMSCs can be differentiated with dexamethasone and β‐glycerophosphate into mineralizing osteoblasts within 2 to 3 weeks, or with dexamethasone, indomethacin, and 3‐isobutyl‐1‐methylxanthine into lipid vesicle–containing adipocytes within 1 to 2 weeks. Phenotypic changes during those highly dynamic differentiation processes can be detected by biochemical and histological assays and gene expression analyses of differentiation markers. In addition, this unit describes an electroporation method that allows the transient genetic perturbation of hMSCs. Curr. Protoc. Stem Cell Biol. 17:1H.3.1‐1H.3.20. © 2011 by John Wiley & Sons, Inc.

Keywords: human mesenchymal stem cell; adipocyte; osteoblast; in vitro differentiation; gene expression analysis; mineralization; lipid vesicle; alkaline phosphatase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation of hMSCs into Osteogenic and Adipogenic Lineages
  • Basic Protocol 2: Transient Transfection of hMSCs by Electroporation
  • Basic Protocol 3: Quantification of Alkaline Phosphatase (ALP) Activity
  • Basic Protocol 4: Quantification of DNA Content
  • Basic Protocol 5: Quantification of Mineralization
  • Basic Protocol 6: Histology and Semi‐Quantification of Mineralization
  • Basic Protocol 7: Histology and Semi‐Quantification of Lipid Vesicles
  • Basic Protocol 8: Gene Expression Analysis of Osteogenic and Adipogenic Differentiation Markers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Differentiation of hMSCs into Osteogenic and Adipogenic Lineages

  Materials
  • hMSC medium (see recipe), prewarmed (37°C)
  • Human bone marrow‐derived mesenchymal stem cells (tested for surface antigens CD105+, CD166+, CD29+, CD44+, CD14, CD34, and CD45; 750,000 cells per vial; expanded to passage 6; e.g., Lonza)
  • 0.4% (w/v) trypan blue (e.g., Invitrogen)
  • Phosphate‐buffered saline (PBS; pH 7.4; e.g., Invitrogen), prewarmed (37°C)
  • 0.05% (w/v) trypsin (e.g., Invitrogen, cat. no. 15400‐054), prewarmed (37°C)
  • Ethanol, absolute (e.g., Fisher Scientific)
  • 1 mM dexamethasone (see recipe)
  • 2 M β‐glycerophosphate (see recipe)
  • 30 mM indomethacin (see recipe)
  • 250 mM 3‐isobutyl‐1‐methylxanthine (IBMX; see recipe)
  • PBS/Triton (see recipe)
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Serological pipets (2‐ml, 5‐ml, 10‐ml, and 25‐ml volume) with motorized serological pipet filler
  • Centrifuge with swing‐out rotor and adaptors for 50 ml “Falcon” tubes
  • Microcentrifuge tubes (0.5 ml volume)
  • Bright‐field microscope
  • Burker glass slide chamber or electric field multichannel cell counting system, e.g., CASY cell counter (Roche Diagnostics)
  • 175‐cm2 cell culture flasks (preferably with angled neck)
  • 12‐well cell culture clusters (22.1 mm diameter) including lids with individual condensation rings
  • Cell scraper (15‐mm, slim blade)
  • Polypropylene cylindrical test tubes (PP tubes; 3 ml, 4 ml or 5 ml volume), or optionally microcentrifuge tubes (2 ml volume)
NOTE: The procedures described in Basic Protocols protocol 11 and protocol 22 are performed in a Class II biological hazard flow hood or a laminar‐flow hood.

Basic Protocol 2: Transient Transfection of hMSCs by Electroporation

  Materials
  • Plasmid DNA (2 µg of expression vector constructs or shRNAi constructs in a concentration of at least 0.4 µg/ml; e.g., Origene, and MISSION shRNA library, Sigma)
  • hMSCs in flasks (confluency of ∼60%; see protocol 1)
  • Human MSC Nucleofector Kit (Lonza)
  • hMSC medium (see recipe), prewarmed (37°C)
  • Amaxa nucleofector system (Lonza) with Amaxa cuvettes (http://www.lonzabio.com/)
  • Additional reagents and equipment for hMSC cell culture (see protocol 1)
NOTE: The procedures described in Basic Protocols protocol 11 and protocol 22 are performed in a Class II biological hazard flow hood or a laminar‐flow hood.

Basic Protocol 3: Quantification of Alkaline Phosphatase (ALP) Activity

  Materials
  • Cellular extracts of interest ( protocol 1)
  • 10 U/ml alkaline phosphatase (from bovine kidney, e.g., Sigma)
  • PBS/Triton (see recipe)
  • p‐nitrophenylphosphate (p‐NPP; see recipe)
  • 0.1 M sodium hydroxide
  • Sonicator (e.g., Soniprep 150, Sanyo)
  • Microcentrifuge tubes (1.5 ml volume)
  • 5‐ml polypropylene cylindrical test tubes (PP tubes)
  • 96‐well clear polystyrene flat‐bottom microtiter plates (e.g., Corning)
  • Microplate reader (with filter for absorbance detection at 405 nm; e.g., Wallac 1420 Victor 2)

Basic Protocol 4: Quantification of DNA Content

  Materials
  • Cellular extracts of interest ( protocol 1)
  • DNA standard (see recipe)
  • Heparin solution (see recipe)
  • RNase solution (see recipe)
  • Ethidium bromide solution (see recipe)
  • Sonicator (e.g., Soniprep 150, Sanyo)
  • 96‐well clear polystyrene flat‐bottom microtiter plates (e.g., Corning)
  • Fluorescence microplate reader (e.g., Wallac 1420 Victor 2)

Basic Protocol 5: Quantification of Mineralization

  Materials
  • Cellular extracts of interest ( protocol 1)
  • 6 M hydrochloric acid
  • PBS/Triton/HCl (see recipe)
  • Calcium chloride standard solution in PBS/Triton/HCl (see recipe)
  • Calcium assay reagent 1 (see recipe)
  • Calcium assay reagent 2 (see recipe)
  • Sonicator (e.g., Soniprep 150, Sanyo)
  • 1.5‐ml microcentrifuge tubes
  • 96‐well clear polystyrene flat‐bottom microtiter plates (e.g., Corning)
  • Microplate reader (with filter for absorbance detection at 595 nm; e.g., Wallac 1420 Victor 2)

Basic Protocol 6: Histology and Semi‐Quantification of Mineralization

  Materials
  • Cultures of hMSC in 12‐well plates ( protocol 1)
  • Phosphate‐buffered saline (PBS; pH 7.4; e.g., Invitrogen), prewarmed (37°C)
  • 10% (v/v) formalin (1:10 dilution of 37% formaldehyde in PBS)
  • Alizarin Red S solution (see recipe)
  • 10% (v/v) acetic acid
  • Ammonium hydroxide
  • Bright‐field microscope with photocamera
  • Plate shaker
  • Cell scraper (15 mm slim blade)
  • 85°C heat block
  • Microcentrifuge for 1.5‐ml microcentrifuge tubes allowing 20,000 × g
  • Microplate reader (with filter for absorbance detection at 405 nm; e.g., Wallac 1420 Victor 2)

Basic Protocol 7: Histology and Semi‐Quantification of Lipid Vesicles

  Materials
  • Cultures of hMSC in 12‐well plates ( protocol 1)
  • Phosphate‐buffered saline (PBS; pH 7.4; e.g., Invitrogen), prewarmed (37°C)
  • 10% (v/v) formalin (1:10 dilution of 37% formaldehyde in PBS)
  • 60% (v/v) 2‐propanol in PBS
  • Oil Red O working solution (see recipe)
  • Igepal working solution: 4% (v/v) Igepal (Sigma) in 2‐propanol
  • Microscope with photocamera
  • Plate shaker
  • 96‐well clear polystyrene flat bottom microtiter plates (e.g., Corning)
  • Microtiter plate reader capable of reading at 490 nm)

Basic Protocol 8: Gene Expression Analysis of Osteogenic and Adipogenic Differentiation Markers

  Materials
  • Cultures of hMSC in 12‐well plates ( protocol 1)
  • Phosphate‐buffered saline (PBS; pH 7.4; e.g., Invitrogen), cold
  • TRIzol (Invitrogen) or equivalent
  • Chloroform
  • 2‐propanol
  • Ethanol, absolute (e.g., Fisher Scientific)
  • 1 M EDTA (RNase‐free; e.g., Invitrogen)
  • 8 M lithium chloride (RNase‐free; e.g., Ambion)
  • 70% ethanol prepared with RNase‐free H 2O
  • RevertAid First Strand cDNA Synthesis Kit (Fermentas)
  • RNase‐and DNase‐free water (e.g., Invitrogen)
  • RT‐PCR master mix stock (see recipe)
  • Primer mix (2.5 µM forward primer + 2.5 µM reverse primer; primer sequences for the detection of osteoblast, adipocyte differentiation and housekeeping genes described in Bruedigam et al., )
  • Cell scraper (15 mm slim blade)
  • Microcentrifuge tubes (0.5 ml and 1.5 ml volume)
  • Refrigerated microcentrifuge allowing 20,000 × g
  • Microvolume spectrophotometer (e.g., NanoDrop)
  • 25°, 42°, 65°, and 70°C water baths or heat blocks
  • 96‐well PCR plates (compatible to thermal cycler system; e.g., Bioplastics)
  • Thermal cycler with fluorescence detection system (excitation wavelength of 495 nm and an emission wavelength of 520 nm; e.g., ABI PRISM 7500 sequence detector, Applied Biosystems)
NOTE: The solutions and equipment used for the RNA isolation and cDNA synthesis techniques described below need to be RNase‐free.
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Figures

Videos

Literature Cited

Literature Cited
   Ali, A.T., Penny, C.B., Paiker, J.E., Psaras, G., Ikram, F., and Crowther, N.J. 2006. The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3‐L1 cells and human preadipocytes. Anal. Biochem. 354:247‐254.
   Bessey, O.A., Lowry, O.H., and Brock, M.J. 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164:321‐329.
   Bieback, K., Kern, S., Kluter, H., and Eichler, H. 2004. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22:625‐634.
   Bruedigam, C., Koedam, M., Chiba, H., Eijken, M., and van Leeuwen, J.P. 2008. Evidence for multiple peroxisome proliferator‐activated receptor gamma transcripts in bone: fine‐tuning by hormonal regulation and mRNA stability. FEBS Lett. 582:1618‐1624.
   Bruedigam, C., Eijken, M., Koedam, M., van de Peppel, J., Drabek, K., Chiba, H., and van Leeuwen, J.P. 2010. A new concept underlying stem cell lineage skewing that explains the detrimental effects of thiazolidinediones on bone. Stem Cells 28:916‐927.
   Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
   Connerty, H.V. and Briggs, A.R. 1966. Determination of serum calcium by means of orthocresolphthalein complexone. Am. J. Clin. Pathol. 45:290‐296.
   Eijken, M., Koedam, M., van Driel, M., Buurman, C.J., Pols, H.A., and van Leeuwen, J.P. 2006. The essential role of glucocorticoids for proper human osteoblast differentiation and matrix mineralization. Mol. Cell. Endocrinol. 248:87‐93.
   Eijken, M., Swagemakers, S., Koedam, M., Steenbergen, C., Derkx, P., Uitterlinden, A.G., van der Spek, P.J., Visser, J.A., de Jong, F.H., Pols, H.A., and van Leeuwen, J.P. 2007. The activin A‐follistatin system: Potent regulator of human extracellular matrix mineralization. FASEB J. 21:2949‐2960.
   Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz‐Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W.C., Largaespada, D.A., and Verfaillie, C.M. 2002. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41‐49.
   Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284:143‐147.
   Wagner, W., Feldmann, R.E. Jr., Seckinger, A., Maurer, M.H., Wein, F., Blake, J., Krause, U., Kalenka, A., Bürgers, H.F., Saffrich, R., Wuchter, P., Kuschinsky, W., and Ho, A.D. 2006. The heterogeneity of human mesenchymal stem cell preparations—evidence from simultaneous analysis of proteomes and transcriptomes. Exp. Hematol. 34:536‐548.
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