2190 Liu Liu et al. Eur. J. Immunol. 2012. 42: 2187–2196

5Institutes of Molecular Medicine and ExperimentalImmunology (IMMEI), University of Bonn,Germany

Acknowledgements: We thank the Housefor Experimental Therapy and the Flow Cy-tometry Core Facility at the Institute ofMolecular Medicine for technical support.This work was supported by the collabo-rative research center SFB645 and grantnumber BU2441/1–1, both funded by theGerman Research Foundation.

Conflict of interest: The authors declareno financial or commercial conflict of in-terest.

References

1 Burgdorf, S. et al., Science 2007. 316: 612–

616.

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2003. 100: 12889–12894.

3 Norbury, C. C. et al., Immunity 1995. 3: 783–

791.

4 Luhrmann, A. and Haas, A., Methods Cell. Sci.

2000. 22: 329–341.

5 Wainszelbaum, M. J. et al., Exp. Cell Res. 2006.

312: 2238–2251.

6 Chavrier, P. et al., Cytometry 1997. 29: 41–49.

7 Guermonprez, P. et al., Nature 2003. 425: 397–

402.

8 Hmama, Z. et al., J. Cell Sci. 2004. 117: 2131–

2140.

9 Bougneres, L. et al., Immunity 2009. 31: 232–

244.

Abbreviations: ERAD: ER-associated degrada-

tion machinery · HEK-MR: MR-expressing

HEK293 T cells · MR: Mannose receptor · SR:

Scavenger receptor

Keywords: Cross presentation � Dendriticcells � Protein trafficking

Full correspondence: Prof. Sven Burgdorf, Lifeand Medical Sciences (LIMES) Institute,University of Bonn, Carl-Troll-Str. 31, 53115BonnFax: +49-228-7362653e-mail: burgdorf@uni-bonn.de

Received: 6/9/2011Revised: 22/3/2012Accepted: 30/4/2012Accepted article online: 15/5/2012

The detailed Materials and methodsfor Technical comments areavailable online in the Supportinginformation

[DOI: 10.1002/eji.201142356]

A novel way to isolateMSCs from umbilicalcords

Mesenchymal stem cells (MSCs) not onlypossess extensive self-renewal potentialand can modulate immune cell activation[1], but also they are easily expanded andstored ex vivo, and are considered to be“immune privileged.” MSCs modulate thefunction of immune cells, including T andB lymphocytes, dendritic cells, and natu-ral killer cells, inhibit their proliferationand alter their cytokine secretion. Previ-ous data from our group and others [2–4]demonstrated the immunosuppressive andantiinflammatory effects of MSCs in thetreatment of several animal disease modelsincluding autoimmune diseases. As a resultof these unique qualities, MSCs are attrac-tive candidates in stem cell-based therapy.

The first successful isolation offibroblast-like colonies from bone marrow,that is, MSCs, was described four decadesago by Friedenstein et al. [5]. The isola-tion method was based on the adherenceof bone marrow-derived, fibroblast-likecells to the plastic cell culture plate,and a concomitant lack of adherenceof bone marrow-derived hematopoieticcells. MSCs separated by adherence haveuniform cell morphology and long-termculture expansion, and the cost of thismethod is low that makes it desirable as apractical method of production.

However, it is reported that MSC sep-aration using the traditional cell-adherentmethod will not yield good results becauseMSCs are rare in tissue; the proportionof MSCs in the bone marrow is less than1:10,000 mononuclear cells [6,7]. There-fore, it is difficult to obtain enough MSCsfrom bone marrow for medical use. In addi-tion, the maternal–fetal interface is an im-

portant source of MSCs, and several groupshave isolated MSCs from umbilical cord,placenta, and decidua [8–10]. Umbilicalcord tissue, which is considered as a clin-ical waste, is the most stable and read-ily available source of MSCs and of worthstudying.

Florian et al. [11] reported that cell iso-lation protocols have a major impact onthe functional activity of bone marrow-derived progenitor cells, highlighting theimportance of optimizing MSC isolationprotocols. There are four methods re-ported to separate MSCs: (i) flow cyto-metrical sorting [12]; (ii) magneticseparation [13]; (iii) density gradientcentrifugation [14]; and (iv) the cell plas-tic adherence method [15,16]. Since thereare no specific surface markers for MSCs,it is difficult to separate MSCs from thedigested cell pellet [17]. Although severalreports describe the use of CD133, CD271,and CD105 as markers to separate MSCs[18–20], this method also requires sev-eral negative markers (CD3, CD14, CD19,CD38, and CD66b) to assist sorting. In ad-dition, flow cytometrical sorting and mag-netic separation have large effects on cellviability and require sophisticated equip-ment. Therefore, these methods are notyet widely used. Density gradient cen-trifugation, on the other hand, also re-quires sophisticated equipment and a suit-able separation medium and is thereforenot suitable for large-scale production ofMSCs.

It should be noted that all the methodsdescribed to isolate MSCs require digestionof the tissue to obtain a single-cell suspen-sion. Since umbilical cords are hard to di-gest by the traditional digestive enzymecollagen, and long-term digestion willaffect the cell viability, there is a dilemmabetween obtaining a single-cell suspensionand obtaining high cell viability.

A few groups also use a tissue explantsadherent method to separate MSCs [21]that seems to avoid this problem. However,the traditional tissue explants adherentmethod has also some serious drawbackssuch as the low adherence of the tissue tothe plastic cell culture plate, and the longtime for separation [21]. Therefore, wecombined the methods of isolating MSCsfrom plastic-adherent tissue and digested

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2012. 42: 2187–2196 Technical comments 2191

Figure 1. Isolation of UC-MSCs from human umbilical cord tissue. (A) Schematic description of the different methods used to isolate UC-MSCs inthis study. (B) Morphology of isolated MSCs by the novel method after 4 days, 6 days, and 8 days in culture (20X). Blue arrow indicates the adherenttissue after using novel method. (C) The cellular growth of UC-MSCs isolated by the novel and traditional methods was determined over 14 days.Data are shown as mean ± SEM of n = 3 samples. (D) The cellular growth curve UC-MSCs was determined at passage 2. Data are shown as themean of n = 3. Data are representative of three independent experiments (B)–(D).

single-cell suspension, using a novel, semi-tissue-adherent method to quickly obtainlarge amount of MSCs from umbilical cord(UC-MSCs). Compared with the traditionalmethod, the number of steps in this novelmethod is less and the total separation timeis short (Fig. 1A).

Using our technique, a mild digestion ofumbilical cords released some MSCs from

the tissue, and also allowed some incom-pletely digested tissue to adhere to plastic;MSCs inside the tissue could then migrateto the plastic and expand (Fig. 1B). Wealso improved the formula of digestiveenzymes, add hyaluronidase and dispasethat prevented unwanted clumping of cellscultured in suspension. Adherent cellswith fibroblastic morphology could be ob-

served as early as 24 h in culture. The cellsformed a monolayer of homogeneous bipo-lar spindle-like cells with a whirlpool-likearray within 1 week of culture (Fig. 1B).

UC-MSCs were then serially passagedto examine their expansion potential anddetermined to expand readily for up to2 weeks (Fig. 1D). We compared thespeed of mesenchymal cell isolation using

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

2192 Liu Liu et al. Eur. J. Immunol. 2012. 42: 2187–2196

Figure 2. Characterization of UC-MSCs derived from human umbilical cord tissue. (A) Flow cytometrical characterization of isolated UC-MSCsduring passage 3. Expression of surface antigens CD105, CD73, CD90, HLA-ABC, CD29, CD44, HLA-DR, CD19, CD11b, CD14, CD34, and CD31was detected using flow cytometry. The percent positive of each marker is shown on the right. Data are representative of three independentexperiments. (B) QPCR analysis of the expression of IL-6, TGF-β, IDO, VEGF, and COX-2. �Ct = (target gene)Ct-U6Ct. Data are shown as mean ±SEM of n = 3 samples and are representative of three independent experiments. (C) The proliferation of T cells cocultured with different ratios ofUC-MSCs, and activated with PHA/IL-2, was assessed by 3H-thymidine incorporation. Data are shown as mean + SEM of n = 3 replicates and arerepresentative of three independent experiments.

our novel method with that of the tradi-tional methods and found that the novelmethod yields MSCs much faster thanthe other two methods (Fig. 1C). Fur-thermore, after three cell passages, theadherent cells were symmetric with phe-notypic surface antigens. The UC-MSCsshared most of their phenotype withbone marrow-derived MSCs (BM-MSCs)as reported, including positivity for CD29,CD44, CD90, CD105 (SH2), CD73 (SH3),and HLA-ABC, negativity for CD19, CD11b,

CD14, CD34, and CD31 (endothelial cellmarker), and HLA-DR (Fig. 2A). BM-MSCssecrete several soluble factors to exercisetheir immunosuppressive properties, suchas IL-6, TGF-β, IDO, VEGF (vascular en-dothelial growth factor), and COX-2 [22].To determine whether UC-MSCs isolatedby our novel method also express thosefactors, we measured mRNA expression ofthese factors. UC-MSCs expressed signifi-cant amounts of VEGF, IL-6, TGF-β, andCOX-2 (Fig. 2B). We also demonstrated

that after 72 h coculture with purifiedhuman T cells, the UC-MSCs inhibitedT-cell proliferation in response to mitogentreatment (Fig. 2C).

In conclusion, we have developed anovel method of isolating MSCs from um-bilical cords for stem cell therapy, whichis much easier and faster than traditionalmethods. The MSCs isolated using ourmethod exhibited the expansion potential,immune phenotype, and immunosuppres-sive properties of BM-MSCs.

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2012. 42: 2187–2196 Technical comments 2193

Liu Liu1, Xiaoyin Zhao1, Pengfei Li1,Guangfeng Zhao2, Yaping Wang1,

Yali Hu2 and Yayi Hou1,3

1Immunology and Reproductive Biology LabMedical School & State Key Laboratory ofPharmaceutical Biotechnology, NanjingUniversity, Nanjing, China

2The affiliated Drum Tower Hospital of Nanjing,University Medical School, Nanjing, China

3Jiangsu Key Laboratory of Molecular Medicine,Nanjing, China

Acknowledgments: This work was sup-ported by the grants from the NationalNatural Science Foundation (project num-ber: 81072410), the Special ResearchGrant of Jiangsu Province Departmentof Health (project number: XK200709and JHB2011–1), and a special grantfor maternal-fetal medicine from Jiangsuprovince health department of China(project number: 81070508).

Conflict of interest: The authors declareno financial or commercial conflict of in-terest.

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Abbreviations: MSC: mesenchymal stem cell ·UC: umbilical cord

Keywords: Isolation � MSC � Umbilical cord

Full correspondence: Dr. Yayi Hou, MedicalSchool, Nanjing University, Hankou Road No.22, Nanjing, Jiangsu, 210093, ChinaFax: +86-25-8368-6441e-mail: yayihou@nju.edu.cn

Additional correspondence: Dr. Yali Hu, Theaffiliated Drum Tower Hospital of NanjingUniversity Medical School, Nanjing, Jiangsu,210093, Chinae-mail: yalihu2009@gmail.com

Received: 27/12/2011Revised: 13/3/2012Accepted: 2/5/2012Accepted article online: 15/5/2012

The detailed Materials and methodsfor Technical comments areavailable online in the Supportinginformation

[DOI: 10.1002/eji.201242436]

High-resolution invivo imaging ofmicroglia using aversatilenongeneticallyencoded marker

Microglial cells are the innate immune cellsof the CNS, whose main role is to mon-

itor the integrity of and to react to anydisturbances of brain homeostasis. As such,microglial cells are involved in a largenumber of CNS insults (e.g. acute CNSinjury, brain tumors, apoptosis, infection,ischemia, neurodegenerative diseases) andtheir engagement can be either neurotoxicor neuroprotective [1,2]. Despite their crit-ical role in ameliorating or exacerbatingdisease progression, little is known aboutthe in vivo functional properties of thesecells.

So far high resolution in vivo stud-ies of microglial function were conductedin mice with genetically labeled microglia[3,4]. These analyses revealed the surveil-lance function of microglia in the healthybrain [5,6], and its involvement in theremodeling of synaptic circuits duringischemia and sensory deprivation [7].However, because of the lack of fractalkinereceptor (CX3CR1 mice) or low expres-sion levels of GFP (Iba1-GFP mice), thesemouse lines are less suitable for studyingthe role of microglia under pathologicalconditions [2,8–10]. Therefore, there is aneed for a nongenetically encoded, easyto use microglial marker, enabling high-quality staining of microglia (similar to thequality obtained in GFP-expressing mice),but applicable to any mouse strain at anyexperimental age.

Here we utilize a well-known his-tological marker tomato lectin (fromLycopersicon esculentum [11–13]) for highresolution in vivo imaging of microglia.A brief pressure injection of tomatolectin conjugated with a fluorescent dyeDyLight R© 594 (TL) into the mouse cor-tex resulted in robust staining of microglialcells (Fig. 1A) and blood vessels (arrow-head). The latter, however, were easily dis-tinguished from microglia based on theirmorphological appearance. A single injec-tion of TL labeled a roughly spherical tis-sue volume with a diameter of 80–130 μm.Multiple, equally spaced injections fromthe same pipette were used to increase thedimensions of the labeled area.

To test the reliability of the in vivostaining protocol, we applied it to theCX3CR1GFP/+ mice previously described[3]. A total of 100% of all GFP-positivecells within the stained area were alsoTL positive (n = 103 cells in four mice;

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu