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International Journal of Biological Macromolecules 54 (2013) 71– 75

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo u r n al hom epa ge: ww w.elsev ier .com/ locate / i jb iomac

ffect of chitooligosaccharides on mice hematopoietic stem/progenitor cells

inlin Wei1, Wei Chen, Fang Mao, Yuanfeng Wang ∗

nstitute of Food Engineering, College of Life & Environment Science, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, PR China

r t i c l e i n f o

rticle history:eceived 26 May 2012eceived in revised form 17 October 2012ccepted 19 October 2012

a b s t r a c t

Chitooligosaccharides (COSs) is a kind of polysaccharide scaffolds used to enhance cartilage repair duringtreatments involving bone marrow stimulation, angiogenesis and osteogenesis increase in vivo. Capabili-ties of COSs in promoting the proliferation and differentiation of hematopoietic stem cells/hematopoietic

vailable online 26 October 2012

eywords:hitooligosaccharidesematopoietic stem cell

progenitor cells were mainly explored in the paper. The results showed that a high concentration of COSssignificantly proliferate the mice marrow cells and induced CD34+ cells into megakaryocyte progeni-tor cells. However, COSs could not enhance the proliferation of CD19+ and CD4+ and promote CD34+cells to differentiate into lymphoid progenitor cells. It suggested that COSs can promote hematopoieticstem/progenitor cells hyperplasia, and the mechanism may be that COSs promote stromal cell secretionof hematopoietic growth factors.

mmunomagnetic beads

. Introduction

Hematopoietic stem cell (HSC)/hematopoietic progenitor cellHPC) possess the unique capacity of multiple differentiation andelf-renewal, and have classically been defined by the presence orbsence of cell-surface antigens [1]. The physical qualities of bloodells mainly rely on proliferation and differentiation of HSC/HPC.plastic anemia patients and cancerous persons those receivedadiotherapy and chemotherapy often suffered from duplex con-usion of immune system and hematopoietic system due to theisease itself or treatment factors. Therefore, it is of importantractical significance to look for a kind of drugs or functionaloods that can regulate both immune and hematopoietic activity.SC/HPCs are originated from immune and hematopoietic systemnd their biological functions increasingly caught the attention ofesearchers. At present, HSC/HPCs are presently being consideredor therapeutic use to prevent hematological, autoimmune diseasend also cardiovascular disease [2,3]. COSs are the degraded prod-cts of chitosan or chitin, which have recently been produced byeveral methods such as enzymatic and acidic hydrolysis. COSs are

kind of polysaccharide biomaterials composed of glucosamineith variable levels of N-acetyl glucosamine. They are biocompat-

ble, cationic and adhesive, biodegradable, and angiogenic whenmplanted in bleeding wounds [4,5]. COSs have becoming the hot

esearch topics due to their biological activities, namely, antimi-robial [6,7], hypocholesterolemic [8,9], drug delivery [10], and

∗ Corresponding author. Tel.: +86 21 27463960.E-mail address: foodlab2000@yahoo.com.cn (Y. Wang).

1 Xinlin Wei contributed equally for this work.

141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2012.10.022

© 2012 Elsevier B.V. All rights reserved.

calcium and ferrum absorption acceleration [11–13], immunity-enhancing and antitumor effects [14–16], and so on.

Immunomagnetic beads activated cell sorting technology wasthat cells are isolated and enriched by connection cells labelingof monoclonal antibody with a special magnetic beads directly orindirectly. This method is fast, economical, and simple [17,18]. Inaddition, it has large work capacity and high purity. Thus, it iswidely used in sorting of the cluster of differentiation (CD) cellsand pre-election enrichment of sub-group. CD molecules can actin numerous ways, often acting as receptors or ligands importantto the HSC/HPC. A signal cascade is usually initiated, altering thebehavior of the cell. Some CD proteins do not play a role in cellsignaling, but have other functions, such as cell adhesion. To ourknowledge, the effect of COSs on HSC/HPC by this method hasnot been reported so far. This work isolated CD34+ cells of mar-row HSC/HPC surface using immunomagnetic beads activated cellsorting technology, labeled CD34+, CD41+, CD19+, CD4+ cells usingimmunofluorescence method, and then determined the quantitiesof positive cells by flow cytometry. The aim of this work was toinvestigate the effects of COSs on cellulae medullares and HSC/HPC,exploring whether COSs could promote proliferation and differen-tiation of HSC/HPC.

2. Materials and methods

2.1. Materials and reagents

Condensing agent, immunomagnetic beads and mouse CD34+antibody were prepared by Professor Li Xingyu (Shanghai NormalUniversity). Healthy ICR mice were purchased from Super – B&Klaboratory animal Corp. MTT, 24-well plates and 96-well plates

7 Biological Macromolecules 54 (2013) 71– 75

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Table 1Effect of chitooligosaccharides with different concentrations on bone marrow.

COS concentration OD570/nm

CK 0.4372 ± 0.00661 mg/ml 0.5022 ± 0.0071*

500 �g/ml 0.4609 ± 0.0027*

50 �g/ml 0.4352 ± 0.01255 �g/ml 0.4384 ± 0.0030

2 X. Wei et al. / International Journal of

ere obtained from Sigma. RPMI 1640 medium, sodium dodecylulfate (SDS), fetal bovine serum (FBS) and CD41+, CD19+, CD4+ere purchased from GIBCO. All other reagents and solvents were

f analytical grade and used without further purification unlesstherwise noted. All aqueous solutions were prepared using newlyouble-distilled water.

.2. Preparations of COSs

COSs were prepared through the hydrolysis of colloidal chitosany the enzyme source obtained from Aspergillus fumigates. 1.0 ml ofnzyme was mixed with 9.0 ml of chitosan (3.0%, w/v). The mixtureas then incubated for 12 h at 50 ◦C. The reaction was stopped by

he addition of 1 ml of trichloroacetic acid (50%) and maintainedor 3 h at 4 ◦C before NaOH solution was added to adjust the pH to.0. Thereafter, the mixture was centrifuged (4500 × g, 15 min) toollect supernatant for obtaining COSs which was further purifiedy Sephadex G-25 chromatography. Identification of it was carriedut as before reported [19].

.3. Separation of mononuclear cells (MNC) from mouse bonearrow

Mice were sacrificed by breaking their necks and immersed inlcohol (75%) for 1 min. The femurs of them were taken out byutting skins of femur and removed of remained muscular tis-ue. Marrow cells were washed with RPMI 1640 medium for 2–3imes to the femur whitish after one hole was drilled in each endf the femur with 1 ml of syringe. The obtained solution was cen-rifuged at 2150 × g for 10 min and the precipitate was collected forbtaining MNC. Finally, cell concentration was adjusted to densityf 1 × 106 cells/ml.

.4. Determination of optimum induction concentration withTT method

The bone marrow was obtained under aseptic condition,hopped and washed through screen mesh (200 meshes) with ster-le normal saline and centrifuged at 2150 × g for 5 min for threeimes. The bone marrow cells precipitates were suspended with

ml of RPMI 1640 medium and seeded at 2 × 106 cell/ml into perell of 96 well plates. Some wells of the cell culture plates were

dded with the COSs sample solution at gradient final concen-rations of 1 mg/ml, 500 �g/ml, 50 �g/ml, 5 �g/ml, 0.5 �g/ml andepeated six times. The cells were incubated at 37 ◦C for 72 h in

humidified atmosphere of 5% CO2 in air. Then 20 �l of MTT5 mg/ml) was added into per well at the 68 h, incubated contin-ously for the rest 4 h prior to be added to 100 �l of 10% SDS andixed thoroughly to dissolve the dark blue crystals. The cell cul-

ure solutions were kept overnight at room temperature. On theext day, the plates were read with an ELISA reader (Austria TECANompany), using test wavelength of 570 nm.

.5. Immunomagnetic beads separation of cells

The prepared magnetic particles were washed in magnetic fieldith physiological saline for twice, added 880 �l physiological

aline and ultrasounded for 5 min. Then 20 �l of mice CD34+ anti-ody were added and incubated for 15 min at 37 ◦C. The incubatedagnetic particles were separated from magnetic field for remov-

ng unreacted material, and washed in physiological saline for 2imes to remove the supernatant. 1 ml of antibiotics were added to

entrifuge tube containing magnetic particles and stored at −20 ◦C.ubsequently, the mouse bone marrow MNC solutions obtainedn 2.3 were mixed with immunomagnetic beads solution with theatio of 1:100 (v/v) at 37 ◦C for 15 min, and the mixture was again

500 ng/ml 0.4359 ± 0.0050

* P < 0.01.

magnetized in magnetic field for another 15 min. At last, RPMI 1640medium was used to wash isolated cells in mixture.

2.6. Co-culture of bone marrow stromal cells with HSC/HPC

Mice bone marrow cells were inoculated in 24-well plates con-taining 20% of FBS and incubated for 10 d to form stromal cell layer,and then the supernatant and non-adherent cells were removed.CD34+ cells were seeded into the 24-well plates at a density of1 × 104 cells/ml and incubated it at 37 ◦C for 4, 7, 11, 14 d in ahumidified atmosphere of 5% CO2 in air before determination byflow cytometry.

2.7. Immunofluorescence mark and flow cytometrydetermination for cells

CD34+ and CD41+ cells were labeled by two kinds of fluorescentdyes, while CD19+ and CD4+ were marked with two other kinds offluorescent dyes. Flow cytometry (BDFACS Calibur Company) wereused to determine quantities of positive cells.

3. Results and discussions

3.1. Preparation of COSs

The average molecular weight of COSs purified by Sephadex G-25 was 1389 Da, the deacetylation degree was 91% determined bytitration (Fig. 1).

3.2. Effect of COSs on mice marrow cells

It is believed that marrow cells are divided into myeloid cellsand lymphatic cells. Myeloid cells include granulocyte mono-nuclear leucocytes, mast cells, dendritic cells, erythrocytes andblood platelet. Lymphatic cells include T cells, B cells, and naturekill cells. The proliferation of these cells indicates that immunologicfunction of organism is enhanced.

The effect of the COSs obtained in this study on mice mar-row cells was studied by measuring the cell numbers using MTTassay. As seen from Table 1, a high concentration of COS (1000 and500 �g/ml) significantly proliferated the mice marrow cells. How-ever, a lower concentration of COS (0.5–50 �g/ml) did not have anyevident proliferation effects on the marrow cells. It is worthy tonote that the COS obtained in this study had no toxicity effect onthe marrow cells because all the absorbance of treatment groupswere not lower than that of blank group (Table 1).

3.3. Effect of COSs on HSC/HPC

As shown in Fig. 2, marrow cells morphology changed withincrease of time. In the first day, no fusiform adherent cells couldbe seen. While three days later, some adherent cells occurred, andin 7–10 d, fusiform cells were the main cells.

X. Wei et al. / International Journal of Biological Macromolecules 54 (2013) 71– 75 73

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Fig. 1. HPLC profiles o

.4. Separation of CD34+ cells by immunomagnetic beads

The isolation, purification and identification of HSC are the firsttep of hematopoietic stem cell research. Nowadays, the usual wayf isolation HSC/HPC is immunomagnetic beads or flow cytom-try method according to differentiation antigen of cell surface.D34+ is the characteristic antigen of HSC/HPC surface. This studyeparated CD34+ cells from mice marrow cell by immunomag-etic beads. This technology is connecting cells (labeled by CD34+)ith special micro beads. It is fast, high purity, easy and sim-le. So it is widely used. This study separated CD34+ cells and

etermined its percentage by flow cytometry. As shown in Fig. 3.he result showed that CD34+ cells accounted for 62.75% of totalells.

Fig. 2. Formation of

me reaction products.

3.5. The influence of COSs on CD34+ and CD41+ cells

CD34+ is the earliest found surface antigen of differentiationof HSC/HPC, which is a breakthrough of hematopoietic stem cellresearch history. The traditional theory believes that screen ofhematopoietic stem cell is mainly dependent on CD34+ cells, andCD34+ is the characteristic surface antigen of HSC/HPC. Referring tothe results of chitooligosaccharides with different concentrationson bone marrow (Table 1), 500 �g/ml, 50 �g/ml, 5 �g/ml COSs ashigh dose group, middle dose group and low dose group, respec-tively. Cells were cultured for 4, 7, 11, 14 d and then analyzed

proliferation effects by flow cytometry. The result showed that theCD34+ and CD41+ cells in COSs treated groups were proliferatedremarkably in contrast to control group (Figs. 4 and 5). The CD34+

stromal cells.

74 X. Wei et al. / International Journal of Biological Macromolecules 54 (2013) 71– 75

Fig. 3. Sorting CD34+ by im

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ig. 4. Effect of chitooligosaccharides with different concentration and actuationuration on CD34 cells.

ells in low concentration COSs group have a significant prolifera-ion but just the opposite for high concentration group. However,igh concentration group and middle concentration group COSshowed a strong stimulation to CD41+ cells. CD41+ is the presented

ig. 5. Effect of chitooligosaccharides with different concentration and actuationuration on CD41 cells.

munomagnetic bead.

the somatic nucleus line cell. The stronger proliferation of CD41+suggested that COSs might stimulate CD34+ into somatic nucleusline cell such as platelet which plays important role in hemokinesis.The proliferation of this type means COSs can provoke CD34+ cellsdifferentiate into megakaryocyte cells in a direct or indirect way.The interaction of cells and COSs occurred in the co-culture systemof stroma cells with HSC/HPC. Therefore, the promotion of COSs oncell proliferation may be due to the promotion of hematopoieticgrowth factors by stroma cells.

3.6. The influence of COSs on CD19+ and CD4+ cells

With the in-depth study of HSC/HPC, scientists found someother characteristic surface antigens except for CD34+. CD19+

and CD4+ also carry out humoral immune and cell immune,respectively. This study marked CD19+ and CD4+ cells usingimmunofluorescence. The flow cytometry result showed thatCD19+ and CD4+ cells in COSs treated groups could not prolifer-

ate, or were even lower than that of control group (Figs. 6 and 7).This proved that COSs could not provoke CD34+ cells differentia-tion to lymphoid progenitor cells. Proliferation and differentiationare the most salient characteristic of HSC.

Fig. 6. Effect of chitooligosaccharides with different concentration and actuationduration on CD4 cells.

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Bioelectronics 26 (2011) 3900–3907.

ig. 7. Effect of chitooligosaccharides with different concentration and actuationuration on CD19 cells.

The primitive HSC can differentiate to two groups: one isyeloid HSC, which differentiates to erythrocyte, granulocyte,onocyte, and blood platelet; the other one is lymphoid stem cell.

his study explored HSC/HPC proliferation and differentiation by ao-culture system of stroma cell and HSC/HPC (COSs were added),hich provided a theoretical reference to new medicinal valueevelopment of COSs.

. Conclusions

In this study, some CD antibodies were used to investigate theffect of COSs on proliferation and differentiation of mice mar-ow cells and HSC/HPC. Low concentration of COSs (5 �g/ml) canromote CD34+ cells proliferation but not for higher concentra-ion in co-culture system. While higher concentration of COSs (50,00 �g/ml) can promote marrow cells proliferation, and which canlso promote CD34+ cells differentiate into megakaryocyte pro-enitor cells, that means COSs has certain regulation function toematopoiesis. However, COSs could not enhance the proliferationf CD19+ and CD4+, which suggested that COSs could not promoteD34+ cells differentiate into lymphoid progenitor cells. Thus, the

ffects of COSs on HSC/HPC mainly induce the proliferation and dif-erentiation of the medullary system HSC. The immunoregulation

echanism of COSs did not depend on HSC/HPC and need furtherxploration.

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ical Macromolecules 54 (2013) 71– 75 75

Acknowledgments

The authors are grateful for financial sponsored by InnovationProgram of Shanghai Municipal Education Creative Commis-sion (12ZZ125, 11ZZ121), Shanghai Rising-Star Tracking Program(11QH1401800), Food Safety and Nutrition Innovation Team ofShanghai Normal University (No. A-3101-12-004003), Develop-ment Center of Plant Germplasm Resources) (No. B-6010-11-001),Produce-learn-Research Project of Shanghai Normal University(No. DCL201207), National High Technology Research and Devel-opment (863) Program of China (2008AA10Z322), National NaturalScience Foundation of China (No. 81072308), Shanghai Biomedicinekey Program (No. 10391901700), and Shanghai Yangtze River DeltaScience Joint Efforts Program (11495810500, 12495810600).

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