Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(2): 173-181 (2017)

Effect of rat cartilage essence on omentum cells

cultured through micromass method

Zahra Goudarzi

, Kazem Parivar

, Susan Kiaei

and Roudabeh Razaz

1,3,4

Master of Biology, Department of Biology, Science and Research Branch, Islamic Azad University,

Tehran, Iran

Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

ABSTRACT

Omentum`s tissue MSCs have recently been recognized as multipotent cells. In this study inducing effects of cartilage

essence obtained from the NMRI bred rat on omentum cells cultured through Micromass method have been evaluated.

12 days NMRI rats were used as cell sample. Omentum tissue lacking fast was removed from the intestines. To culture

in cellular compressed mass through micromass, cells numbers were added in level unit. Before culturing, cells were

precipitated at the bottom of the tube like a compressed mass by centrifuge and then DMEMculturing environment

containing glutamine, glucose and sodium pyruvate, without sodium B carbon was added to the resulted cellular

mass. Furthermore, FBS serum amount was reduced to 2% in culturing environment gradually. Thereafter, stem cells

were exposed to various doses of cartilage essence. Investigations showed that density rate of cartilage essence 50

Landa were able to ease the process of turning to cartilage for these cells in 21 days. Cartilage essence`s other densi-

ties have led to the elimination of stem cells. To prove the differentiation of mesenchymal cells chondroblast turn,

Alison blue and Toluidine blue which are the speci c colors of outer cellular matrix was used.

KEY WORDS: MESENCHYMAL CELLS, OMENTUM TISSUE, STEM CELLS, CARTILAGE ESSENCE, CHONDROBLAST, DIFFERENTIATION

173

ARTICLE INFORMATION:

*Corresponding Author:

Received 12

April, 2017

Accepted after revision 28

June, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

174 EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Zahra Goudarzi et al.

INTRODUCTION

Stem cells are a kind of an undifferentiated somatic cell

which have two unique characteristics: Self-renewal:

which they can continuously replicate themselves in

culturing environment and maintain their restoration

for an in nite period. Differentiation: which they can

under certain conditions turn into speci ed cells and

differentiate other constructing cell type’s pf a living

body. These cells exist in blood, bone marrow, skeletal

muscle, retinal, retina, teeth pulp, brain, nerve cord,

liver, skin, intestine, pancreas and venous blood (Kadi-

var, M., 2009). Regarding replication and differentiation

potential, stem cells with different sources are different

in a way that this potential decreases from embryonic

stem cells to adult’s stem cells (Baksh, 2004).

Despite embryonic stem cells high potential, their use

due to problems related transplant rejection of the cells,

ethical problems and tumorigenicity of these cells after

transplant is highly limited (Romanov, 2003). Unlike

embryonic stem cells, MSCs are not replicated perma-

nently whereas after some time face senescence. Studies

showed that cell replication in lab environment leads to

the decrease of their differentiating replication ability

process (Baqb an et al. 2008). Till today stem cells func-

tions included genomics studies, biological processes

studies and development of cell treatment. Currently

scientists use stem cells for screening of new medicines

in labs. (Toyooka, 2003). Methods of identifying primary

MSCs are diverse. Flowcytometry is based on the MSCs

reaction to a number of antibodies (Alagumuthu, 2006,

Chapel, 2003, Liu,2010).

Previous researches have proved the  exibility of

stem cells differentiated from nerve cell, skin cover, lung,

liver, intestine, kidney, and spleen. (Evans, 2002, Mar-

tin 2003). Studies showed that MSCs, when cultured in

a 22 monolayer fashion, show lesser Chondrogenic dif-

ferentiation compared to 3d culturing. We believe that

cell when culturing in 3D fashion (cellular mass) experi-

ences a similar environment to precartilage environment

in embryonic genesis period (Johnstone,1998). Under

these condition chondrocytes maintain their cartilage

phenotype. It seems that the main reason of cartilage

phenotype maintenance in micromass culturing is the

cellular reaction due to direct cell to cell contact in cel-

lular mass. Omentum is a big peritoneal wrinkle. From

omentum`s special features we can refer to its rich source

of angiogenic factor, having a strong lymphatic system,

having the ability to produce security specialized cells

and a big source of various growth factors such as neu-

rotransmitters, neurotrophic factors and in ammatory

mediators. Furthermore, it includes multipotent stem

cells which can differentiate between various types of

cells (Carlos 1992, Khaluqi, 2011).

Various ways can be considered for creating directed

differentiation in lab environment which can include

adding growth factor(s) or chemical morphogens (Li,

wg; 2005), co-culturing of stem cells with inducing

cells, stem cells transplant to certain regions or organs

and etc. In this research project cartilage essence effect

on omentum MSCs differentiation toward cartilage cells

in micromass culturing with gradual reduction of the

serum is investigated.

MATERIAL AND METHODS

In this research 12 days NMRI bred rat, ranging between

18-22 gms, were used as test animals.All the dilutions

used in this article wereprovided though commerce and

were used without further puri cation. Purchased cul-

turing environment was DMEM (Dulbeccos Modi ed

Eagles Medium-high glucose), containing glutamine,

glucose sodium pyruvate, without sodium bicarbonate

from Royan Stem Cells technology Co. FBS dilution

which contains required proteins and factors for cell

growth, was produced from cow serum. PBS without

calcium and magnesium ions was provided with its PH

domain within the range of 7.2 to 7.4. All the dilutions

used in culturing such as PBS, DMEM, and FBS were

sterile before use by passing through syringe head  lter

with 0.22 micrometer penetrative diameter.

First under the laminar hood wish disinfected using

UV and alcohol 10ml PBS dilution was poured into 10cm

Petri dishes. Then, a couple of pen strep drops (penicillin

and streptomycin dilution) were added. In animal room

a rat was made unconscious and was transferred to the

surgery bed. Using 70 & alcohol all of its body especially

its stomach surface was disinfected and stomach`s wall

and peritoneum were exposed using scissors and stretch-

ing stomach to the sides. Then,using forceps and scissors

omentum tissue lacking fat was removedfrom intestines.

Omentum tissues placed on ice were washed using cool

PBS dilution containing 1% penstrep three times so that

it became blood cell free, cellular wastes and any other

possible pollution free. Thereafter, omentum tissues were

gathered in a new Petri dish containing sterile DMEM,

using scissors fragmented and then centrifuged.

Common technique for chondrogenic differentiation

of MSCs in lab environment through micromass method

was derived from Johnstone et al., method (Johnstone

et al.,1998). In this technique, to simulate differentia-

tion environment in a natural way in embryo, a greater

number of cells were exposed to the essence in falcon in

a compressed fashion in a way that 250000 to 300000

in 500 landa of culturing environment were added using

centrifuge with 1200 rpm for 5 minutes in a compressed

fashion slowly at the bottom of the falcon. Also, FBS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED 175

Zahra Goudarzi et al.

serums amount in culturing environment was reduced

gradually. Meaning when changing culturing environ-

ment, each time a lesser amount of FBS were added to

DMEM. Therefore, cells were cultured with 2% FBS

Almost 24 hours after the birth of rat, their stemum

cartilage tissue was removed and were homogenized and

centrifuged. We discard the above dilution and a speci c

amount of nitrogen was added tp cartilage parts depos-

ited, inside a rocky pounder. Due to sudden reduction of

cartilage parts temperature, in its matrix of mid-cellular

tissue crystals are formed which leads to easy fragmen-

tation by the pounder. We added 4ml DMEM containing

FBS15% to the easily fragmented cartilage parts. Now

we homogenize them with high rmp to obtain a suspen-

sion of cartilage. It will take 5 to 7 minutes. Then we

transfer to centrifuge and centrifuge it with 700rpm. We

remove the above dilution and  lter it with 0.45 microm-

eter syringe head  lter. Dilution withdrawn is cartilage

essence. Five groups were studied. Respectively, observer

which only contained culturing environment with 10%

serum, free group or without serum and falcons with

various doses, with doses of 20, 40, and 50 landa which

serum amount in each of the gradually was reduced to

2% and placed in incubator for 21 days.

Densities for obtaining 1000l or 1cc culturing envi-

ronment are as follows in thistest. (For 1cc differentiat-

ing material prepared, 0.1ml, pen/strep is added to the

environment).

• Observer (10%FBS + 90%DMEM)1000µl

• Environment without serum (100%DMEM) 1000 µl

• 20 landa dose 20µl (cartilage essence) +(2%

FBS + 98%DMEM)980µl

• 40 landa dose 40µl (cartilage essence) +(2%

FBS + 98%DMEM)960µl

• 50 landa dose 50µl (cartilage essence) +(2%

FBS + 98%DMEM)950µl

To prove chondroblast differentiation of mesenchymal

cells we use Alison Blue and Toluidine Blue coloring

which are the speci c colors of outer cellular matrix. To

color differentiated cells with Alison blue and Toluidine

bluw,  rst cells were  xed with formaldehyde  xator. To

color with toluidine blue, after consolidation, watering

with alcohol and clari cation with xylene, are molded

by paraf n and 5 micrometer cuts are taken from them.

Toluidine blue is treated to cellular plates for 30 seconds

in room temperature with 50 landa cartilage essence and

in the end extra color is washed off with distilled water.

To color with Alison blue, 0.1 Alison blue color is

diluted in 10ml PBS with the help of PHmeter and add-

ing normal hydrochloric acid 0.01 its PH is determined

in the range of 6 to 6/5. Thereafter it was added to the

cellular plated treated with 50 landa cartilage essence

and were incubated for an approximately one hour.

In the end we discarded the dilution on the cells and

washed them with PBS dilution and scanned them. To

prove the existence of chondroblast cells with immu-

nohistochemistry test after tissue consolidation and

preparation, molding with paraf n was performed. After

providing levels, clari cation and watering, levels con-

solidation was performed by sten. To neutralize peroxide

activity of inner tissue hydrogen, samples were placed in

3 percent hydrogen peroxide for 30 minutes. To reveals

antigens, pepsin enzyme with the amount of 1mg in 1ml

acetic acid 0.5m for 40 minutes in 37cc degree was used.

Thereafter samples were incubated in primary antibodies

against collagen molecules of rat type II for 24 hours in

4cc degree. In the next stage, secondary antibody con-

nected to polymer containing horseRadish peroxidase,

were added to the samples and after washing them with

PBS buffer, di-amino benzidie chromogen was added to

the samples. To color the background, hematoxylin was

used. After washing the samples with buffer, study was

conducted by microscope.

RESULTS AND DISCUSSION

One of the ways of identi cation and assertion of mes-

enchymal nature of cells is using cell surface markers.

Flowcytometry analysis of CD45 is negative and for

CD44 and CD90 is positive. Therefore, mesenchymal

nature of created cells from omentum is con rmed.

PERCENT OF MARKERS

Omentum: A) CD90 Antibody in 84.5 percent of the

analyzed cells is expressed. B) CD45 antibody which

only in approximately 1.92 percent of analyzed cells is

expressed. C) CD44 in 88.22 percent of the cells were

expressed.

FIGURE 1. Flow cytometry  gure for omentum cul-

tured cells.

176 EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Zahra Goudarzi et al.

FIGURE 2. Flow cytometry analysis for cultured cells.

In the current study RNA samples were extracted

from omentum cultured cells and inverted transcription

reaction was performed using designed starters. In this

test, we used WT-1 gene as the marker of mature stem

cells and Oct-4 as the marker of embryonic powerful

cells, and ACTB gene as the house keeper (this gene is

always expressed, if not it is an indication of incorrect

use of PCR). Observations showed that in this test both

WT-1 and Oct-4 genes are expressed, however, RT-PCR

band related to MRNA with WT-1 gene was brighter and

clearer than RT-PCR band related to mRNA with Oct-4

gene. Therefore, WT-1 and Oct-4 gene expressions are

proof of stem cells existence in omentum.

Cellular mass enlargement shows that cells discharge

matrix among themselves and created cartilage tissue.

To prove this claim, after the end of 21 days of differen-

tiation, we performed sectioning on the samples. Inves-

tigations was carries out by microscope and colored by

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED 177

Zahra Goudarzi et al.

Image 1. RT-PCR test results for speci c genes expression of omentum stem cells. Cellular plates were

measured every two days which are as follows:

1. Observer: cellular plate decomposed and disappeared after one week.

2. Environment without serum: cellular plate shrink and decomposed and disappeared after

one week.

3. 20 landa dose: till the  rst week it was observer sized, however gradually after one week

decomposed and disappeared.

4. 40 landa dose: It gew in the  rst week and became bigger than the observer. However, in

the second week cellular mass shrink and decomposed and disappeared.

5. 50 landa dose: cellular plate became bigger compared the previous in the  rst week and

reached more than 1

IMAGE 2. Images are related to omentum mesenchymal cells cultured through 3D culturing

method after cartilage effect with the amount oof 2% FBS serum. a) 20 landa dose of cartilage

essence after one week. b) 40 landa dose of cartilage essence after one week.

Alison blue and Toluidine blue and Collagen 2 expres-

sion was investigated by immunohistochemistry.

Microscopic investigations showed that with serum

reduction better differentiation occurs and chondrocytes

affected by the cartilage essence with 50 landa dose

showed better morphology. Following images con rm

these claims.

Colored sections with toluidine blue show metachro-

matic characteristic and background material turns into

purple.

178 EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Zahra Goudarzi et al.

IMAGE 3. Images related to cultured omentum mesenchymal cells through 3D culturing method

after the cartilage essence effect with 50 landa dose and amount of 2% FBS. a) After one day

which as big as observer group. b) After one week which is slightly bigger.

IMAGE 4. Images related omentum cultured msenchymal cells with 3D culturing method after

cartilage essence effect with 50 landa dose and 2% FBS amount. a) After two weeks. b) After

three weeks.

IMAGE 5. Image is related to omentum mesenchymal cells 21 days after the cartilage essence effect with 50

landa dose and 2% FBS. a) Dual and ternary cells are completely visible in clear lacuna halo. b) Cells differentia-

tion are completely obvious.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED 179

Zahra Goudarzi et al.

IMAGE 6. Image is related to 3D culturing section 21 days after cartilage essence effect with 50 landa dose

and 2% FBS serum. After coloring with toluidine blue, cells round morphology and their dark core is visible

in image b.

IMAGE 7. Images are related to omentum mesenchymal cells 21 days after the

cartilage essence effect. Green colors (a,b) are collagen 2 molecules indicator and

green (c,d) are related to cores coloring by IP.

In coloring with Alison bluematerial  nds a bluish

green background. Image 6: Image is related to the DC

culturing section 21 days after cartilage essence effect

with 50 landa dose and 2% FBS serum. After color-

ing with Alison blue, a community of cells which that

have turned into a colony and discharged matrix among

themselves is seen. To investigate the expression of Col-

lagen type 2, primary and secondary antibodies which

are anti- collagen type 2 are used in the cartilage dif-

ferentiated cells, which the results are visible in the fol-

lowing images.

Stem cells are a kind of undifferentiated somatic cells

that are found in the majority of the multicellular liv-

ings. Some of their characteristics are self-renewal abil-

ity, high mitotic distribution and differentiation power

toward different types of speci ed cells (Becker, 1963,

Siminovitch, 1963). We can refer to MSCs as one of the

most important mature stem cells that have attracted

researcher’s attention. Omentum mesenchymal cells play

a role in treating damages and it was identi ed in 1948

by Cannaday for the  rst time (Kadivar, 2009). These

cells are utilized as supporting cells for inner growing

organs in vivo conditions (Iwashima, 2011). Recently

extracting MSCs from omentum tissue is carried out

successfully (Alagumuthu, 2006, Singh, 2008). In this

study too,  rst omentum mesenchymal cells of 12 days

180 EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Zahra Goudarzi et al.

NMRI ratis extracted and summarized. Some lab stud-

ies have shown that if mature chondrocytes are cultured

in simple dishes, lose their differentiation rapidly and

their physiological activities decrease (Ng 2005). One

of the important conditions for creating culturing is

using micromass (Johnstone,1998). Weused this method

for creating suitable cellular mass, which is increasing

number of cells in level unit.

Thereafter, cells phenotype and genotype was con-

 rmed with various evaluations such as  ow cytometry.

Accordingly, in human and other rodents such as rat, sur-

face antigens related to these cells were investigated by

 ow cytometry and using monoclonal antibodies. What

today is important in identi cation of MSCs, is if they

have  broblast morphology and are negative in terms

of hematopoietic stem cells, they can be presented as

MSCs (Barry, 2004). Expressing speci c markers of MSCs

includes CD90 and CD44 and lack of expressing special

marker of hematopoietic cells is con rmed as CD45 in

separated cells from the omentum, which is aligned with

Pancholi et.al results (Pancholi et al 2010 Li, 2005).

In this research cartilage essence was used for cre-

ating differentiation induction instead of using com-

mon growth factors, since using growth factors such as

TGF can have unknown effects on other cells and have

tumorigenic effects. However, using natural essences in

addition tolacking the above mentioned shortcomings

can have the advantage of lesser transplant rejection due

to tissue similarities and cells differentiate with more

similar conditions with their natural den. Treatment

with cellular essence, is a new strategy for cells differ-

entiation which is able to transform a type of somatic

cell into another. MSCs affected by cartilage essence

induction can  nd chondrocyte cells morphology. For

instance cells took an oval shape which is a characteris-

tic of chondrocyte cells (Shamblott, 1998). Then matrix

construction begins to separate chondroblasts. Differen-

tiation occurs from the center toward exterior. There-

fore, more central cells have chondrocytes characteris-

tics, however, ambient cells typically are chondroblasts

(Liu, 2010).

Various dilutions of cartilage essence were used, after

mesenchymal cells were treated by cartilage essence

with 50 landar dose, signi cant changesoccurred from

the fourth day onward. In some regions cellular colonies

were created. With an increase in number of cells, they

absorb cellular extras. And cells move toward oval and

round formation, which means they are chondroblasts

which later use of speci c Alison blue and Toludine blue

coloring con rmed this notion. These cells seem com-

pletely alive and active and replication process still con-

tinues in them. These change occur while in other groups

under study, their cells still remain between chondro-

cytes morphology phase and spindly and lengthy mor-

phology which is speci c tp  broblasts and with passage

of time face senescence and apoptosis.

Collagen type 2 is the most abundant existing col-

lagen in cartilage. Collagen type 2 contains speci c

sequences to connect to the cell and as a result has

a high interaction with the cell. It can be suitable for

maintaining cartilage cells phenotype and activities (Li,

2005, Zambrano, et al, 1982). In this experience we were

able to con rm production of collagen type 2 through

immunohistochemistry in cartilage cells derived from

omentum MSCs of NMRI breed rat after 21 days from

the start of differentiation induction. Therefore, consid-

ering cellular mass and morphological changes in the

cultured stem cells it seems that we succeeded in pro-

ducing chondroblasts.

CONCLUSION

This research is carried out to investigate inducing

effects of cartilage essence obtained from rat on cul-

turing omentum cells through micromass with reducing

serum amount and understanding and interpreting the

results. Results are the indication of the cartilage essence

effect on omentum`s MSCs in the absence of growth fac-

tors, common induction and their differentiation with

chondrocytes. It seems that our method of separating

omentum`s mesenchymal cells and producing cellular

lnie was completely successful and powerful and have

appropriate and acceptable progress toward differentia-

tion, specially creating a compression and production of

chondroblasts. Considering the results that Dubler et.al

have obtained from culturing on collagen and results

that Tuli and Okafor obtained regarding culturing with

tissue engineering using collagen type 2, we can con-

clude that the cartilage essence used in this research as

an induction factor can intensify differentiation process

in cells due to existence if collagen in itself with the help

of creating and maintain cellular connections.

REFERENCES

Alagumuthu, M., Das, BB., Pattanayak, SP., Rasananda, M.,

2006, The omentum: A unique organ of exceptional versatility,

Indian J Surg; 68:136-41.

Baksh, D., Song, L., Tuan, R.S, 2004, Adult mesenchymal stem

cells: Characterization, differentiation and application in cell

and gene therapy, J. cell. mol. Med, vol l8, No 3, 301-316

Baqban Eslami Nejad, M, Talkhabi, M, Zeynali, B, Eftekhari

Yazdi, P, 2008. Investigating Lithium chloride on replication

rate of MSCs derived from rat`s bone marrow. Year 6. Issue 23.

Pages 363-372

Barack Johnstone, T.M. Hering, A.I. Caplan etal.,1998 Invitro

chondrogenesis of bone marrow-derived mesenchymol pro-

genitor cells, Exp Cell Res, Vol. 238, no.1, PP. 265-272,

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF RAT CARTILAGE ESSENCE ON OMENTUM CELLS CULTURED 181

Zahra Goudarzi et al.

Barry, F-P-, Murphy, J.M., 2004, Mesenchymal stem cells:

clinical application and biological characterization, The

international journal of biochemistry & cell Biology 36:568-

584

Becker, A.J., Mcculloch, E.A., Till, J.E., 1963, Cytological dem-

onstration of the clonal nature of spleen colonies derived from

transplanted rat marrow cells, Nature; 197:452-4

Carlos Junquera, L, jose carneiro, Robert. O.K: (1992), Basic

histology

Chapel, A., Bertho, JM., Bensidhoum, M., Fouillard, L.,Young,

R.G., 2003, Mesenchymal stem cells home to injured tissues

when co-infused with hematopoietic cells to treat a radiation–

induced multi-organ failure syndrome, J Gene Med; 5(12):

1028-38

Evans, MG., Kaufman, MH., Establishment in culture of Pluari

potential Cell from rat embryos. Nature (2002) 2, 2 : 154-156

Gomez, I., 2012, Omentum in the Repair of Injured Tissue:

Evidence for Omental Stem Cells,stem Cells and Cancer Stem

Cells, Volume 2

Iwashima, M., Love, R., Braun, R., Sethupathi, P., Knight, K.L.,

2011, Omentum as a source of stromal/ stem cells and medical

treatments using stroma) stem cells, Patent Application pub-

lication

Jones, EA S.E. Kinsey, A. English et al., Isolation and Charac-

terization of bone marrow multipotential mesenchymol pro-

genitor cells, Arthritis Rheum, Vol. 46, no.12, PP. 3349-3360,

2002

Kadivar, M. Piryiayi F and Ramezani, M. 2009. Separation,

culturing and MSCs differentiation of chickens bone marrow.

Journal of Armaqan Danesh. Round 14. Issue 4

Khaluqi, K. Shahbazi, A. Sojoudi, M. Vosouq, M. 2011. Stem

cells in simple language. Tehran. House of Biology. First Vol-

ume

Li, WG Tuli, R; Okafor, C A three –dimensional nano brous

scaffold for cartilage tissue engineering using human mesen-

chymal steem cells Biomaterials, (2005)

Liu, Y., Yue, W., Ji, L., Nan, X., Pei, X., 2010, Production of

erythriod cells from human embryonic stem cells by fetal liver

cell extract treatment, BMC Developmental Biology; 10:85

Martin GR. 2003 Isolation of a Pluripotent Cell line From early

rat embryos cultured in medium conditioned by teratocarci-

noma Stem cells . Proc . Natl . Acad . Sci, 78: 7634-7638

Ng, S., Wua, Y.N., Zhou, Y., Tohb, Y.E., Ho, Z.Z., Chia, S.M.,

2005, Optimization of 3D hepatocyte culture by controlling the

physical and chemical properties of the extra- cellular matri-

ces, Biomaterials; 26(16) :3153-3163

Pancholi, N., Patel, J., Gudehitblu, K., Kraus, M., Dunea, G.,

Arruda, J., Singh, AK., 2010, Culture of omentum-induced

regenerating liver yielded hepatocyte-committed stem cells,

Tranalation Research, Voiume l56, Number 6; 156(6):358-68

Pournasr, B. Baharvand, H. 2010. Stem cells and human`s msen-

chymal: biological characteristics, treatment functions and sep-

aration methods. Tehran. House of Biology. Volume 2. 28-67

Romanov, YA., Swvintsitskaya, VA., Smirnov, VN., Searching

for alternative Sources of postnatal humar mesenchymal Stem

cells: candidate MSC like cells from umbilical cord . Stem Cells

(2003) 21 : 105-110

Shamblott, MJ., Axelmanj, J., 1998 Derivation of pluripotent

stem cells from Cultured human primordial germ cells. Proc

Natl Acad Sci, (1998).

Siminovitch, L., Mcculloch, E.A., Till, J.E., 1963, The distribu-

tion of colony-forming cells among spleen colonies. Journal of

cellular and comparative physiology; 62:327-36

Singh, A.K., Patel, G., Litbarg, N.O., Gudehithlu, H.P.,

Sethupathi, P., Arruda, G.A . and Dunea, G., 2008, Stromal

cells cultured from omentum express pluripotent markers, pro-

duce high amount of VEGF and engraft to injured sites. Cell

and Tissue Research, 332:81-88

Toyooka, Y; Tsunekawa, N; Akasu, R; 2003 Embryonic stem

cells can from germcells in vitro.

Zambrano, NZ; et al. collagen arrangement in cartilages Acta

Anat (1982). 113:26