Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation

Mahmood S Choudhery, Michael Badowski, Angela Muise, John Pierce, David T Harris, Mahmood S Choudhery, Michael Badowski, Angela Muise, John Pierce, David T Harris

Abstract

Background: Human adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism's reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs).

Methods: To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30 years), adult (35-50 years) and aged (>60 years) individuals were investigated. MSCs were characterized for expression of the genes p16(INK4a) and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential.

Results: Aged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs.

Conclusions: In conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies.

Figures

Figure 1
Figure 1
Comparison of age related parameters in AT-MSCs isolated from young and aged donors. (A, B) Gene profiling of AT-MSCs indicate that gene expression of p16 and p21 is higher is AT-MSCs isolated from aged as compared to young donors. Data is shown as fold-induction as compared to untreated control cells. (C, D) Similarly, aged cultures show senescent features as determined by SA-β-gal staining compared to young cultures (data shown as percent positive cells). Concomitant with these features young AT-MSCs have higher level of SOD (E; data shown as absorbance values) and viability after stress (F; data shown as percent viable cells). Results are expressed as Mean ± Standard deviation. *P < 0.01 for young AT-MSCs versus aged AT-MSCs. AT-MSCs: adipose tissue derived mesenchymal stem cells, SA-β-gal: senescence associated beta galactosidase.
Figure 2
Figure 2
Yield and growth characteristics of MSCs. Cells per gram of adipose tissue decreases with increased age of the donors (A). CFU assay was performed to enumerate number of cells in SVF that can form colonies. Number of CFUs deceases with age of the donor (B). Proliferative potential is associated with donor age as indicated by number and time for population doublings. Number of population doublings of MSCs decreases (C) while time per population doubling increases (D) with age of the donor. Results are expressed as mean ± standard deviation. MSCs: mesenchymal stem cells, CFU, colony forming unit assay. *P < 0.01 for young AT-MSCs versus aged AT-MSCs.
Figure 3
Figure 3
Adipogenic potential of AT-MSCs is independent of donor age. Adipogenic differentiation was carried out for AT-MSCs isolated from young, adult and aged individuals. Insets show MSCs cultured in normal expansion medium. Adipogenic experiments were terminated after 21 days and oil red O was used to stain for lipid rich vacuoles, as shown (Figure 3A-C). The percentage of cells that stained positive for oil red O was determined, followed by quantification of oil red O uptake. Adipogenic differentiated MSCs isolated from young (A), adult (B) and aged (C) donors stained positive for oil red O. Differentiation levels varied between groups but non-significantly as indicated by counting oil red O positive cells (D) and colorimetrically by evaluating oil red O uptake (E). Adipogenic differentiation was further confirmed through real time RT-PCR and a non-significant difference was found when different age groups were compared (F). Results are expressed as mean ± standard deviation.
Figure 4
Figure 4
Effect of donor age on osteogenic potential of AT-MSCs. Osteogenic induction was assessed by von Kossa staining. (A-C) Representative figures showing matrix mineralization in induced cultures of young, adult and aged, respectively. (D-F) Control AT-MSCs did not stain positive with von Kossa staining. Possible age related differences in osteogenic potential were measured using ImageJ software. AT-MSCs isolated from young individuals revealed more matrix mineralization than adult and aged groups (G). Similar, results were obtained when gene expression of OST and ALP was analyzed through quantitative RT-PCR (H). Results are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 for young AT-MSCs versus aged AT-MSCs, #P for adult versus aged AT-MSCs. OST: osteocalcin, ALP: Alkaline phosphatase.
Figure 5
Figure 5
Chondrogenic potential of AT-MSCs is compromised by donor age. Differentiation of AT-MSCs was induced chondrogenic induction medium for 21 days in micromass pellet culture. Alcian blue staining was performed for induced AT-MSCs isolated from (A) young, (B) adult and (C) aged individuals. At higher microscopic magnification (shown in insets) the cartilage like tissue appeared to be composed of rounded cells, surrounded by lacunae lying in a proteoglycan rich extracellular matrix. Chondrogenic in vitro potential was quantified by a colorimetric assay in which Alcian blue uptake (blue color) was extracted with 6 M guanidine HCl and absorbance was read at 620 nm. (D1) showing micromass 30 minutes after incubation and (D2) after 120 minutes. (E) absorbance values were significantly higher for young and adult AT-MSCs compared to aged. (F) Quantitative RT-PCR was performed for mRNA expression of aggrecan and collagen type 2. Age related decline in mRNA level was observed for both genes. Results are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 for young AT-MSCs versus aged AT-MSCs, #P for adult versus aged AT-MSCs.
Figure 6
Figure 6
The neuron-cell-like morphology of AT-MSCs isolated from young, adult and aged donors. (A-C) Neuron-like-morphology was observed in MSCs isolated from young, adult and aged individuals, respectively. (D) Percentage of AT-MSCs showing neuron-like morphology. Representative slide showing nestin expression as determined by immunofluorescence staining (E) with similar expression in all groups (F). Real time RT-PCR analysis showed equivalent up-regulation of neurogenic specific genes (NFM and NSE) in AT-MSCs isolated from donors of different age (G). NSE: Neuron-Specific-Enolase, NFM: Neurofilament.

References

    1. Pérez-Simon JA, López-Villar O, Andreu EJ, Rifón J, Muntion S, Campelo MD, Sánchez-Guijo FM, Martinez C, Valcarcel D, Cañizo CD. Mesenchymal stem cells expanded in vitro with human serum for the treatment of acute and chronic graft-versus-host disease: results of a phase I/II clinical trial. Haematologica. 2011;96(7):1072–1076. doi: 10.3324/haematol.2010.038356.
    1. Choudhery MS, Khan M, Mahmood R, Mohsin S, Akhtar S, Ali F, Khan SN, Riazuddin S. Mesenchymal stem cells conditioned with glucose depletion augments their ability to repair -infarcted myocardium. J Cell Mol Med. 2012;16(10):2518–2529. doi: 10.1111/j.1582-4934.2012.01568.x.
    1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–147. doi: 10.1126/science.284.5411.143.
    1. Lee CC, Ye F, Tarantal AF. Comparison of growth and differentiation of fetal and adult rhesus monkey mesenchymal stem cells. Stem Cells Dev. 2006;15(2):209–220. doi: 10.1089/scd.2006.15.209.
    1. Choudhery MS, Badowski M, Muise A, Harris DT. Comparison of human adipose and cord tissue derived mesenchymal stem cells. Cytotherapy. 2013;15(3):330–343. doi: 10.1016/j.jcyt.2012.11.010.
    1. Ryan JM, Barry F, Murphy JM, Mahon BP. Interferon-γ does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol. 2007;149(2):353–363. doi: 10.1111/j.1365-2249.2007.03422.x.
    1. Abumaree M, Al Jumah M, Pace RA, Kalionis B. Immunosuppressive properties of mesenchymal stem cells. Stem Cell Rev. 2012;8(2):375–392. doi: 10.1007/s12015-011-9312-0.
    1. Amado LC, Saliaris AP, Schuleri KH, St John M, Xie JS, Cattaneo S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005;102(32):11474–11479. doi: 10.1073/pnas.0504388102.
    1. Xin H, Li Y, Shen LH, Liu X, Wang X, Zhang J, Pourabdollah-Nejad DS, Zhang C, Zhang L, Jiang H, Zhang ZG, Chopp M. Increasing tPA activity in astrocytes induced by multipotent mesenchymal stromal cells facilitate neurite outgrowth after stroke in the mouse. PLoS One. 2010;5(2):e9027. doi: 10.1371/journal.pone.0009027.
    1. Taylor SE, Smith RK, Clegg PD. Mesenchymal stem cell therapy in equine musculoskeletal disease: scientific fact or clinical fiction? Equine Vet J. 2007;39(2):172–180. doi: 10.2746/042516407X180868.
    1. Kretlow JD, Jin YQ, Liu W, Zhang WJ, Hong TH, Zhou G, Baggett LS, Mikos AG, Cao Y. Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol. 2008;9:60. doi: 10.1186/1471-2121-9-60.
    1. Choudhery MS, Khan M, Mahmood R, Mehmood A, Khan SN, Riazuddin S. Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation and anti-apoptosis capabilities. Cell Biol Int. 2012;36(8):747–753. doi: 10.1042/CBI20110183.
    1. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13(12):4279–4295. doi: 10.1091/mbc.E02-02-0105.
    1. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multi-lineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211–228. doi: 10.1089/107632701300062859.
    1. Zhu M, Kohan E, Bradley J, Hedrick M, Benhaim P, Zuk P. The effect of age on osteogenic, adipogenic and proliferative potential of female adipose-derived stem cells. J Tissue Eng Regen Med. 2009;3(4):290–301. doi: 10.1002/term.165.
    1. Giovannini S, Diaz-Romero J, Aigner T, Heini P, Mainil-Varlet P, Nesic D. Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro. Eur Cell Mater. 2010;20:245–259.
    1. Kim WK, Jung H, Kim DH, Kim EY, Chung JW, Cho YS, Park SG, Park BC, Ko Y, Bae KH, Lee SC. Regulation of adipogenic differentiation by LAR tyrosine phosphatase in human mesenchymal stem cells and 3T3-L1 preadipoctes. J Cell Sci. 2009;122(pt 22):4160–4167.
    1. Nalesso G, Sherwood J, Bertrand J, Pap T, Ramachandran M, De Bari C, Pitzalis C, Dell’accio F. WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways. J Cell Biol. 2011;193(3):551–564. doi: 10.1083/jcb.201011051.
    1. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop DJ, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells: the international society for cellular therapy position statement. Cytotherapy. 2006;8(4):315–317. doi: 10.1080/14653240600855905.
    1. Alt EU, Senst C, Murthy SN, Slakey DP, Dupin CL, Chaffin AE, Kadowitz PJ, Izadpanah R. Aging alters tissue resident mesenchymal stem cell properties. Stem Cell Res. 2012;8(2):215–225. doi: 10.1016/j.scr.2011.11.002.
    1. Capparelli C, Chiavarina B, Whitaker-Menezes D, Pestell TG, Pestell RG, Hulit J, Andò S, Howell A, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neo-angiogenesis. Cell Cycle. 2012;11(19):3599–3610. doi: 10.4161/cc.21884.
    1. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92(20):9363–9367. doi: 10.1073/pnas.92.20.9363.
    1. Menicanin D, Bartold PM, Zannettino AC, Gronthos S. Genomic profiling of mesenchymal stem cells. Stem Cell Rev Rep. 2009;5(1):36–50. doi: 10.1007/s12015-009-9056-2.
    1. Stolzing A, Jones E, McGonagle D, Scutt A. Age-related changes in human bone marrow derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev. 2008;129(3):163–173. doi: 10.1016/j.mad.2007.12.002.
    1. Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, Zandiehdoulabi B, Schouten TE, Kuik DJ, Ritt MJ, Van Milligen FJ. Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies. Cell Tissue Res. 2008;332(3):415–426. doi: 10.1007/s00441-007-0555-7.
    1. Gonzalez R, Griparic L, Vargas V, Burgee K, Santacruz P, Anderson R, Schiewe M, Silva F, Patel A. A putative mesenchymal stem cells population isolated from adult human testes. Biochem Biophys Res Commun. 2009;385(4):570–575. doi: 10.1016/j.bbrc.2009.05.103.
    1. Erickson GR, Gimble JM, Franklin DM, Rice HE, Awad H, Guilak F. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun. 2002;290(2):763–769. doi: 10.1006/bbrc.2001.6270.
    1. Jang S, Cho HH, Cho YB, Park JS, Jeong HS. Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin. BMC Cell Biol. 2010;11:25. doi: 10.1186/1471-2121-11-25.
    1. Dimmeler S, Leri A. Aging and disease as modifiers of efficacy of cell therapy. Circ Res. 2008;102(11):1319–1330. doi: 10.1161/CIRCRESAHA.108.175943.
    1. Scheubel RJ, Zorn H, Silber RE, Kuss O, Morawietz H, Holtz J, Simm A. Age-dependent depression in circulating endothelial progenitor cells in patients undergoing coronary artery bypass grafting. J Am Coll Cardiol. 2003;42(12):2073–2080. doi: 10.1016/j.jacc.2003.07.025.
    1. Tokalov SV, Grüner S, Schindler S, Wolf G, Baumann M, Abolmaali N. Age-related changes in the frequency of mesenchymal stem cells in the bone marrow of rats. Stem Cells Dev. 2003;16(3):439–446.
    1. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol. 1999;107(2):275–281. doi: 10.1046/j.1365-2141.1999.01715.x.
    1. Robinson JA, Harris SA, Riggs BL, Spelsberg TC. Estrogen regulation of human osteoblastic cell proliferation and differentiation. Endocrinology. 1997;138(7):2919–2917.
    1. Ankrom MA, Patterson JA, d’Avis PY, Vetter UK, Blackman MR, Sponseller PD, Tayback M, Robey PG, Shapiro JR, Fedarko NS. Age-related changes in human oestrogen receptor α function and levels in steoblasts. Biochem J. 1998;333(Pt 3):787–794.
    1. Shi YY, Nacamuli RP, Salim A, Longaker MT. The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging. Plast Reconstr Surg. 2005;116(6):1686–1696. doi: 10.1097/01.prs.0000185606.03222.a9.
    1. Khan WS, Adesida AB, Tew SR, Andrew JG, Hardingham TE. The epitope characterisation and the osteogenic differentiation potential of human fat pad-derived stem cells is maintained with ageing in later life. Injury. 2009;40(2):150–157. doi: 10.1016/j.injury.2008.05.029.
    1. Murphy JM, Dixon K, Beck S, Fabian D, Feldman A, Barry F. Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis Rheum. 2002;46(3):704–713. doi: 10.1002/art.10118.

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