The nutritional requirements of stem cells have not been determined; in particular, the amino acid metabolism of stem cells is largely unknown. In this study, we investigated the amino acid metabolism of human mesenchymal stem cells (hMSCs), with focus on two questions: Which amino acids are consumed and/or secreted by hMSCs and at what rates? To answer these questions, hMSCs were cultured on tissue culture plastic and in a bioreactor, and their amino acid profile was analyzed. The results showed that the kinetics of hMSCs growth and amino acid metabolism were significantly higher for hMSCs in tissue culture plastic than in the bioreactor. Despite differences in culture conditions, 8 essential and 6 nonessential amino acids were consumed by hMSCs in both tissue culture plastic and bioreactor cultures. Glutamine was the most consumed amino acid with significantly higher rates than for any other amino acid. The metabolism of nonessential amino acids by hMSCs deviated significantly from that of other cell lines. The secretion of alanine, glycine, glutamate, and ornithine by hMSCs showed that there is a strong overflow metabolism that can be due to the high concentrations of amino acids provided in the medium. In addition, the data showed that there is a metabolic pattern for proliferating hMSCs, which can contribute to the design of medium without animal serum for stem cells. Further, this study shows how to implement amino acid rates and metabolic principles in three-dimensional stem cell biology.
Get full access to this article
View all access options for this article.
References
1.
MastersJ.R., StaceyG.N.Changing medium and passaging cell lines. Nature Protocols, 2:2276. 2007.
2.
EagleH.Nutrition needs of mammalian cells in tissue culture. Science, 122:501. 1955.
3.
SimpsonN.H., SinghR.P., PeraniA., GoldenzonC., Al-RubeaiM.In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene. Biotechnol Bioeng, 59:90. 1998.
4.
KontoravdiC., WongD., LamC., LeeY.Y., YapM.G.S., PistikopoulosE.N.et al.Modeling amino acid metabolism in mammalian cells toward the development of a model library. Biotechnol Prog, 23:1261. 2007.
5.
DuvalD., DemangelC., MunierjolainK., MiossecS., GeahelI.Factors controlling cell-proliferation and antibody-production in mouse hybridoma cells .1. Influence of the amino-acid supply. Biotechnol Bioeng, 38:561. 1991.
6.
SanfeliuA., CairoJ.J., CasesC., SolaC., GodiaF.Analysis of nutritional factors and physical conditions affecting growth and monoclonal antibody production of the hybridoma KB-26.5 cell line. Biotechnol Prog, 12:209. 1996.
7.
AlbertsB., JohnsonA., LewisJ., RaffM., RobertsK., WalterP.Molecular Biology of the Cell, 5th. New York: Garland Science, 2008.
8.
ChoiK.M., YoonH.H., SeoY.K., SongK.Y., KwonS.Y., LeeH.S.et al.Effect of essential and nonessential amino acid compositions on the in vitro behavior of human mesenchymal stem cells. Korean J Chem Eng, 24:1058. 2007.
9.
CaplanA.Why are MSCs therapeutic? New data: new insight. J Pathol, 217:318. 2009.
10.
ProckopD.J.Marrow stromal cells for nonhematopoietic tissues. Science, 276:71. 1997.
11.
OwenM., FriedensteinA.J.Stromal stem-cells—marrow-derived osteogenic precursors. Ciba Foundation Symp, 136:42. 1988.
LarsonB.L., YlostaloJ., ProckopD.J.Human multipotent stromal cells undergo sharp transition from division to development in culture. Stem Cells, 26:193. 2008.
21.
GregoryC.A., GunnW.G., ReyesE., SmolarzA.J., MunozJ., SpeesJ.L.et al.How wnt signaling affects bone repair by mesenchymal stem cells from the bone marrow. Stem Cell Biol Dev Plast, 1049:97. 2005.
22.
SantosF.D., AndradeP.Z., BouraJ.S., AbecasisM.M., SilvaC.L.D., CabralJ.M.S.Ex Vivo expansion of human mesenchymal stem cells: a more effective cell proliferation kinetics and metabolism under hypoxia. J Cell Physiol, 223:27. 2010.
23.
HigueraG., SchopD., JanssenF., van Dijkhuizen-RadersmaR., van BoxtelT., van BlitterswijkC.A.Quantifying in vitro growth and metabolism kinetics of human mesenchymal stem cells using a mathematical model. Tissue Eng Part A, 15:2653. 2009.
24.
SekiyaI., LarsonB.L., SmithJ.R., PochampallyR., CuiJ.-G., ProckopD.J.Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells, 20:530. 2002.
25.
ColterD.C., ClassR., DiGirolamoC.M., ProckopD.J.Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A, 97:3113. 2000.
26.
ColterD.C., SekiyaI., ProckopD.J.Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A, 98:7841. 2001.
27.
FrauenschuhS., ReichmannE., IboldY., GoetzP.M., SittingerM., RingeJ.A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Biotechnol Prog, 23:187. 2007.
28.
MartinI., WendtD., HebererM.The role of bioreactors in tissue engineering. Trends Biotechnol, 22:80. 2004.
29.
YangY., RossiF.M.V., PutninsE.E.Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin culture. Biomaterials, 28:3110. 2007.
30.
SchopD., JanssenF.W., BorgartE., de BruijnJ.D., van Dijkhuizen-RadersmaR.Expansion of mesenchymal stem cells using a microcarrier based cultivation system: growth and metabolism. J Tissue Eng Regen Med, 2:126. 2008.
SchopD., van Dijkhuizen-RadersmaR., BorgartE., JanssenF.W., RozemullerH., PrinsH.J.et al.Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism. J Tissue Eng Regen Med, 4:131. 2010.
33.
BothS.K., van der MuijsenbergA.J., van BlitterswijkC.A., de BoerJ., de BruijnJ.D.A rapid and efficient method for expansion of human mesenchymal stem cells. Tissue Eng, 13:3. 2007.
34.
KallinowskiF., RunkelS., FortmeyerH.P., ForsterH., VaupelP.L-Glutamine: a major substrate for tumor cells in vivo?J Cancer Res Clin Oncol, 113:209. 1987.
35.
SchopD., JanssenF.W., van RijnL.D., FernandesH., dev BruijnJ.D., van Dijkhuizen-RadersmaR.Growth, metabolism and growth inhibitors of mesenchymal stem cells. Tissue Eng Part A, 15:1877. 2009.
36.
ZhaoF., ChellaR., MaT.Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: experiments and hydrodynamic modeling. Biotechnol Bioeng, 96:584. 2007.
37.
van ’tRietK., TramperJ.Basic Bioreactor Design, 1st. New York: Basel: Marcel Dekker, Inc., 1991.
38.
MeinelL., KarageorgiouV., FajardoR., SnyderB., Shinde-PatilV., ZichnerL.et al.Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng, 32:112. 2004.
39.
WeiJ., HeoS.J., KimD.H., KimS.E., HyunY.T., ShinJ.W.Comparison of physical, chemical and cellular responses to nano- and micro-sized calcium silicate/poly(epsilon-caprolactone) bioactive composites. J R Soc Interface, 5:617. 2008.
40.
AlmeidaA., BolanosJ.P., MoncadaS.E3 Ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation. Proc Natl Acad Sci U S A, 107:738. 2010.
41.
QuesneyS., MarcA., GerdilC., GimenezC., MarvelJ., RichardY.et al.Kinetics and metabolic specificities of Vero cells in bioreactor cultures with serum-free medium. Cytotechnology, 42:1. 2003.
42.
WahlA., SidorenkoY., DaunerM., GenzelY., ReichlU.Metabolic flux model for an anchorage-dependent MDCK cell line: characteristic growth phases and minimum substrate consumption flux distribution. Biotechnol Bioeng, 101:135. 2008.
43.
NelsonD., CoxM.Lehninger Principles of Biochemistry. New York: Worth Publishers, 2000.
44.
ZhangP.C., McGrathB.C., ReinertJ., OlsenD.S., LeiL., GillS.et al.The GCN2 eIF2 alpha kinase is required for adaptation to amino acid deprivation in mice. Mol Cell Biol, 22:6681. 2002.
45.
ReesW.D.Manipulating the sulfur amino acid content of the early diet and its implications for long-term health. Proc Nutr Soc, 61:71. 2002.
46.
WangJ., AlexanderP., WuL.J., HammerR., CleaverO., McKnightS.L.Dependence of mouse embryonic stem cells on threonine catabolism. Science, 325:435. 2009.
47.
BendallS.C., HughesC., StewartM.H., DobleB., BhatiaM., LajoieG.A.Prevention of amino acid conversion in SILAC experiments with embryonic stem cells. Mol Cell Proteomics, 7:1587. 2008.
48.
DrewsM., DoverskogM., OhmanL., ChapmanB.E., JacobssonU., KuchelP.W.et al.Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR. J Biotechnol, 78:23. 2000.
49.
TritschG.L., MooreG.E.Spontaneous decomposition of glutamine in cell culture media. Exp Cell Res, 28:360. 1962.
ZhouS., CuiZ., UrbanJ.P.Nutrient gradients in engineered cartilage: metabolic kinetics measurement and mass transfer modeling. Biotechnol Bioeng, 101:408. 2008.
52.
GurkanU.A., AkkusO.The mechanical environment of bone marrow: a review. Ann Biomed Eng, 36:1978. 2008.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
