research article characterization and expression of

Research ArticleCharacterization and Expression of Senescence Marker inProlonged Passages of Rat Bone Marrow-Derived MesenchymalStem Cells

Noridzzaida Ridzuan12 Akram Al Abbar3 Wai Kien Yip4

Maryam Maqbool12 and Rajesh Ramasamy12

1Stem Cell amp Immunity Group Immunology Laboratory Department of Pathology Faculty of Medicine and Health SciencesUniversiti Putra Malaysia 43400 Serdang Malaysia2Regenerative Medicine Research Program Genetic and Regenerative Medicine Research CentreFaculty of Medicine and Health Sciences Universiti Putra Malaysia 43400 Serdang Malaysia3Clinical Genetics Unit Department of Obstetrics amp Gynecology Faculty of Medicine and Health SciencesUniversiti Putra Malaysia 43400 Serdang Malaysia4Immunology Unit Department of Pathology Faculty of Medicine and Health Sciences Universiti Putra Malaysia43400 Serdang Malaysia

Correspondence should be addressed to Rajesh Ramasamy rajeshupmedumy

Received 1 February 2016 Revised 23 May 2016 Accepted 23 May 2016

Academic Editor Franca Fagioli

Copyright copy 2016 Noridzzaida Ridzuan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study is aimed at optimizing the in vitro culture protocol for generation of rat bone marrow- (BM-) derivedmesenchymal stem cells (MSCs) and characterizing the culture-mediated cellular senescence The initial phase of generation andcharacterization was conducted using the adherent cells from Sprague Dawley (SD) ratrsquos BM via morphological analysis growthkinetics colony forming unit capacity immunophenotyping and mesodermal lineage differentiation Mesenchymal stem cellswere successfully generated and characterized as delineated by the expressions of CD901 CD44H CD29 and CD71 and lack ofCD11bc and CD45 markers Upon induction rBM-MSCs differentiated into osteocytes and adipocytes and expressed osteocytesand adipocytes genes However a decline in cell growth was observed at passage 4 onwards and it was further deciphered throughapoptosis cell cycle and senescence assays Despite the enhanced cell viability at later passages (P4-5) the expression of senescencemarker 120573-galactosidase was significantly increased at passage 5 Furthermore the cell cycle analysis has confirmed the in vitroculture-mediated cellular senescence where cells were arrested at the G0G1 phase of cell cycle Although the currently optimizedprotocols had successfully yielded rBM-MSCs the culture-mediated cellular senescence limits the growth of rBM-MSCs and itspotential use in rat-based MSC research

1 Introduction

Mesenchymal stem cells (MSCs) were originally isolated byFriedenstein and colleagues in guinea pigrsquos bone marrow [1]characterized as multipotent adult stem cells that grow as fociwith fibroblast-like morphology termed as colony formingunit-fibroblasts (CFU-f) The early exploration and evalua-tion of MSCrsquos biology were mainly achieved by exploitingbone marrow (BM) as a major source To date the common

and efficient source of MSCs is remained to be the bonemarrow Although the frequency of MSCs within the bonemarrow is very low (0001ndash01 of mononuclear cells) MSCsare capable of extensive proliferation and expansion at in vitroculture [2]

It is now recognized thatMSCs reside inmany tissues andorgans other than bonemarrow [3] such as adipose tissue [4]liver [5] skeletalmuscle [6] umbilical cord [7] umbilical cordblood [8] placenta [9] and synovium [10] Besides human

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 8487264 14 pageshttpdxdoiorg10115520168487264

2 Stem Cells International

Table 1 Basal media and FBS concentration used for CFU-f assay

Basal media FBS concentration ()Roswell Park Memorial Institute (RPMI) with GLUTAMAX-I 10RPMI with GLUTAMAX-I 20Dulbeccorsquos Modified Eaglersquos Medium with nutrient mixture F12 (HAM) [1 1] DMEMF12) with GLUTAMAX-I 10DMEMF12 with GLUTAMAX-I 20Low glucose Dulbeccorsquos Modified Eaglersquos Medium (LDMEM) with GLUTAMAX-I 10LDMEM with GLUTAMAX-I 20High glucose Dulbeccorsquos Modified Eagle Medium (HDMEM) with GLUTAMAX-I 10HDMEM with GLUTAMAX-I 20

MSCs have also been isolated from several animal speciesnamely mouse [11ndash13] baboon [14] rat [15 16] monkey[13] rabbit [17] dog [18] cat [19] and horse [20] Despitethe initial understanding on targetedlimited differentiationtowardsmesodermal lineages the current studies have shownthat under a right stimulation MSCs are able to differentiateinto cells of endodermal and ectodermal origins such as 120573-pancreatic cells [21] hepatocyte [22] cardiomyocyte [23]skeletal muscle [24] neuron cells [25] and epithelial cells[26]

Many types of research that test the in vivo nature orfunctions of MSCs are still remarkably relying on animalmodels Employing animals in biological science has becomeindispensable as most of the pharmacological and toxico-logical studies have been developed using laboratory-basedanimals as testing tools For instance the convenience ofmicemodel with a possible genetic modification and availabilityof research reagents and species-specific antibodies ease thepreclinical studies [27 28] Beside mouse model some largerscale studies and disease-specificmodels were also developedusing another type of animal such as rat rabbit fish pigand monkey [29ndash33] In regard to current MSC researchhuman and mouse MSCs are extensively used in the presentresearch scenario as these MSCs are relatively easy to beharvested and expanded at in vitro culture [34 35] Whenconcerning other species it has been reported that the ratMSCs are difficult to isolate and culture-expanded at the invitro culture [15 36 37] This conundrum drives the stemcells research using rat MSCs which is not as lucrative andwell embraced as mouse counterpart However there is aneed for the rat-based MSCs especially in diabetic researchwhere most of the animal model investigations are tailoredbased on rats [38 39] One of the major concerns whicharise in rat MSCs culture is to maintain a stable in vitroculture that provides an uninterrupted supply of stem cellsfor ongoing laboratory and animalworksNumerous researchstudies have reported the paucities of rBM-MSC culturewhere after certain rounds of passages the cells were failed tobe amplified at adequate numbers Despite this observationthe exact mechanism of such growth retardation is not fullyelucidated Thus this research project is aimed at optimizingthe laboratory protocol for isolation characterization andexpansion of rat MSC to fill the gap in the current rat-basedMSCs studies

2 Materials and Methods

21 Animals Sprague Dawley (SD) rats (250ndash350 g and 5ndash8 weeks) were obtained from Chenur Supplier (KajangSelangor Malaysia) Animals were acclimatized and handledwith standard animal care procedures as prescribed byInstitutional Animal Care and Use Committee UniversitiPutra Malaysia (IACUC UPM) Animals were sacrificed bycervical dislocation

22 Generation of Rat Bone Marrow MSCs Bone marrowcells were obtained from Sprague Dawley (SD) rats byflushing femurs and tibiae and cultured in 25 cm2 flask in acomplete culture medium with 1 penicillinstreptomycin05 Fungizone and 01 gentamycin (Gibco United King-dom) After 72 hours of plating nonadherent cells wereremoved and medium was replaced at every 48 hoursAdherent cells were further propagated and upon reaching80ndash90 confluency cells were trypsinised by using 005trypsin-EDTA (Gibco United Kingdom) at 37∘C for 3ndash5minutes Harvested cells were cultured in 25 cm2 flasks forfurther expansion During expansion period media werechanged every 2 days Expanded cells were either used fordownstream experiments or cryopreserved using freezingmedia (10 DMSO and 90 FBS) Media used in rBM-MSC expansion were LDMEM with GLUTAMAX-I (GibcoUnited Kingdom) supplemented with 20 foetal bovineserum (FBS) 1 penicillinstreptomycin 05 Fungizoneand 01 gentamycin (GibcoUnitedKingdom) Supplementsused in the optimization were 20 ngmL basic fibroblastgrowth factor (bFGF) (RampD System USA) 1 nonessentialamino acids (NEAA) (Gibco United Kingdom) and 1insulin transferrin sodium selenite (ITS) (Sigma AldrichUSA)

23 Colony Forming Unit-Fibroblast Assay Colony formingunit-fibroblast (CFU-f) assay was conducted with 1 times 106of nucleated cells from freshly isolated rat bone marrowand seeded in 60mm2 cell culture dish (Becton DickinsonUSA) for 10 days Various basal media were consumed toassess CFU-f as presented in Table 1 and purchased fromGibco United Kingdom Complete media were constitutedwith an individual basalmedium supplementedwith 1peni-cillinstreptomycin 05 Fungizone and 01 gentamycin

Stem Cells International 3

Table 2 Primer sequence for adipocyte and osteocytes

Primer 51015840-31015840 Sequence Amplicon size Annealing temperature (∘C)

GAPDH ForwardReverse

TGAACGGGAAGCTCACTGGTCCACCACCCTGTTGCTGTA 360 481

Osteopontin ForwardReverse

CCGATGAATCTGATGAGTCCTTTCCAGCTGACTTGACTCATGG 303 578

Osteonectin ForwardReverse

ATGAGGGCCTGGATCTTCTTTCTCAAAGAAGTGGCAGGAAGAGTCGA 372 605

PPAR120574 ForwardReverse

GCCTTGCTGTGGGGATGTCTCGAAACTGGCACCCTTGAAAAAT 354 473

CEBPA ForwardReverse

GCAGAAGGTGTTGGAGTTGAAGCGACCCTAAACCATCCTC 214 667

Media change was conducted every 2 days of interval Thecells were stained with 15 crystal violet (Sigma AldrichUSA) in 100 methanol and incubated for 10 minutes Thenumber of colonies more than 2mm was considered andcounted

24 Immunophenotyping The expression of cell surfacemarkers was measured by a direct immunofluorescencestaining and analysed by flow cytometer Cells at passages2-3 were trypsinised and cell count was performed usingtrypan blue exclusion test Upon staining cells were trans-ferred into Fluorescence Activated Cell Sorting (FACS) tubesand washed with 1x phosphate buffer saline (1xPBS) Cellswere labelled with fluorochrome conjugated mouse anti-rat antibodies (CD901-PE CD45-PE CD11BC-PE CD29-FITC CD71-FITC and CD44H-FITC) for 15 minutes at 4∘CFor analysis 10000 cells were acquired by LSR Fortessa flowcytometer (BD Biosciences USA) and analysed using FACSDiva Software (BD Biosciences USA)

25 Differentiation Assays The adipocyte and osteogenic dif-ferentiation capabilities of passages 2-3 expanded rBM-MSCswere performed using StemPro adipogenesis differentiationkit (Gibco Invitrogen USA) and StemPro osteogenesis dif-ferentiation kit (Gibco Invitrogen USA) respectively withminor modifications Rat bone marrow mesenchymal stemcells were cultured in 60mm2 Petri dish and incubated at37∘C in 5 CO2 humidified air Upon reaching 100 conflu-ency cells were supplemented with respective differentiationmedium where the inductive medium was changed every 2days for 20 days For adipogenic induction cells were fixedin 4 paraformaldehyde and stained with Oil Red O solutionwhereas for osteogenic differentiation cells were fixed in icedcold 70 ethanol and stained with Alizarin Red solution

26 Reverse Transcription Polymerase Chain Reaction (RT-PCR) Cellular differentiation of rBM-MSCs towards meso-dermal lineages was further confirmed with gene expressionassay using RT-PCR Total RNA of cells that were culturedin either normal medium or induction media (adipo andosteo) retrieved using TRIzol reagent kit (Invitrogen USA)DNase treatmentwas carried out by adding 205120583LRNAwith

Table 3 PCR gene amplification conditions for GAPDH osteo-pontin osteonectin PPAR120574 and CEBPA gene for adipogenesis andosteogenesis differentiation assays

Process Temperature (∘C) Time CyclePredenaturation 95 3min 1Denaturation 95 30 sec 35Annealing 1min 35GAPDH 481Osteopontin 578Osteonectin 605PPAR120574 473CEBPA 667

Elongation 72 1min 35Final elongation 72 10min 1Incubation 10 infin infin

25 120583L NE buffered DNase 1 1 120583L of RNase inhibitor and 1 120583LDNase 1 and incubated for 30 minutes at 37∘C Subsequently05 120583L of 025M ethylene diamine tetraacetic acid (EDTA)was added and incubated for 10 minutes at 75∘C The RNAconcentration was determined by using spectrophotometerThe cDNA was obtained by reverse transcription of 4 120583gof total RNA using 1 120583L oligo dT primer Primers for PCRwere adopted from the literature [16 18 40ndash42] as shownin Table 2 The detailed PCR procedures for cDNA werefollowed per Table 3

27 Tritiated Thymidine Incorporation Assay The prolifer-ation of rBM-MSCs was determined by tritiated thymi-dine (3H-TdR) assay (Perkin Elmer USA) The radioactivenucleotide 3H-TdR integrates only into actively proliferatingcells during DNA synthesis and the amount of 3H-TdRmeasured is directly proportional to the cell proliferationFive thousand cells per well were cultured in 96-well platefor 24 h 48 h and 72 h Cultures were pulsed with 10 120583L3H-TdR (05 120583Ciwell) at 24 h prior to measurement At theend of each time point (24 h 48 h and 72 h) cells wereharvested onto glass filter mat (Perkin Elmer USA) by usinga 96-well plate automated cell harvester (Harvester Mach


III M TOMTEC USA) The filter mat was dried usingoven (Penasonic Malaysia) (120∘C) for 10min before adding5mL scintillation fluid (OptiPhase SupermixCocktail PerkinElmer USA) The filter mat was then sealed and fitted intoa scintillation cassette for radioactive measurement usingluminescent Microbeta counter (MicroBetaTrilux) Resultswere expressed as count per minute (CPM)

28 Growth Kinetic and Doubling Time Growth kinetic ofrBM-MSCs was performed using trypan blue exclusion testApproximately 150000 cells were plated into 6-well plateCells were grown for 6 days with medium change whichwas performed at every 3 days Cells were harvested everyday and counted Growth kinetic curves of rBM-MSCs fromdifferent passages were plotted The initial seeding numberdays in culture and yield of MSC cultures were recorded andcomputed into doubling timeDoubling timewas determinedby Patterson Formula 1 and expressed asmean doubling time

119879119889 =119879 log 2

log (1198731199051198730) (lowast)

where 119879119889 is the doubling time (h) 119879 is the time during whichcells proliferated from1198730 to119873119905 (h) and119873 is the cell count

29 Apoptosis Assay Apoptosis assay was performed usingAnnexin VDead Cell Apoptosis kit with FITC conjugatedAnnexin V and PI (Invitrogen USA) Annexin V is Ca2+-dependent phospholipid binding protein that binds to phos-pholipid such as phosphatidylserine (PS) Annexin V alongwith propidium iodide (PI) allows identification of earlyapoptotic cells (PI negative FITCAnnexinVpositive) Viablecells with intact membranes exclude PI whereas membranesof dead and damage cells are permeable to PI [43] Approx-imately 100000 cells were washed with 1x Annexin bindingbuffer (ABB) and stained with 2120583L Annexin V and 1 120583LPI for 15min at room temperature Cells were resuspendedin 500120583L 1xABB and acquired using LSR Fortessa flowcytometer (BD Bioscience USA) For analysis 10000 cellswere acquired and analysed using FACS Diva Software

210 Senescence Associated 120573-Galactosidase (SA 120573-gal)Staining Acid 120573-D-galactosidase is a hydrolase localizedin lysosome which cleaves 120573-linked terminal galactosylresidues from substrates such as gangliosides glycoproteinsand glycosaminoglycans [43] Mammalian cells expressedlysosomal120573-galactosidase (120573-gal) activity at pH 40Meanwhile SA 120573-gal activity can be detected at pH 6in senescence cells but not in proliferating cells Bothlysosomal 120573-gal and SA 120573-gal can be detected in situ bya cytochemical staining using chromogenic substrate 5-bromo-4-chloro-3-indolyl 120573-D-galactopyranoside (X-gal)[44] X-gal cleaved by 120573-gal to produce insoluble blueprecipitates [45] Cells at passage two and passage five werefixed with 70 ethanol for 10 minutes and stained with X-galin its respective buffer (pH 4 and pH 6) overnight beforeviewing under the phase contrast microscope Senescentcells were evidenced by the formation of blue precipitatewithin the cells

211 Cell Cycle Analysis Cell cycle analysis of rBM-MSC wasdetermined by measuring DNA content using PI dye Cells atpassage 2 and passage 5 were cultured in 25 cm2 flasks Uponreaching 80ndash90 confluency cells were harvested and fixedwith 70 ethanol and subjected to overnight incubation atminus20∘C Fixed cells were washed with 1xPBS and incubatedwith 05mL staining buffer which consisted of 100120583gmL PI(Molecular Probe Invitrogen) and 20 ngmL RNase (Sigma)in 1xPBS for 30minutes At least 10000 cells were acquired byflow cytometer and analysed via ModFit LT software (VeritySoftware House USA)

212 Statistical Analysis Values for all measurements werepresented asmean plusmn SD unless otherwise stated Comparisonwas performed by Studentrsquos t-test and one-way ANOVASignificance levels were set at value 119901 le 005

3 Results

31 Optimization of rBM-MSC Culture Upon in vitro cul-ture single cells of rat BM have started to form adherent cellcolonies from day 3 onwards The colony of spindle-shapedcells has profoundly increased in size at day 5 and day 7(Figure 1(a)) To determine the optimal media for the growthof rBM-MSCs several basal media and two concentrationsof FBS were tested for the ability to support the growth ofcolony forming unit-fibroblast and cell expansion Figure 1(b)shows the stained CFU-f of LDMEM HDMEM RPMIand DMEMF12 basal media supplemented with 10 FBSor 20 FBS respectively Regardless of the types of basalmedia 20 supplemented FBS yields the highest number ofcolonies as compared to 10 FBS Among all basal mediaLDMEM reaps the highest number of colonies (CFU-f =52) followed by DMEMF12 (CFU-f = 26) RPMI (CFU-f= 24) and HDMEM (CFU-f = 12) (Figure 1(c)) To verifywhether the number of colonies formed is accompanied bythe total cell numbers BM cells from passage 0 were culturedin respective basal media and serum concentrations Thenumber of expanding cells was calculated using trypan blueexclusion test at stipulated time points As evidenced in CFU-f assay the total cell counts are greater when 20 of FBS wasconsumed whereas in terms of the type of basal mediumLDMEM induced a higher cell proliferation as compared toHDMEM RPMI and DMEMF12 (Figure 1(d))

32 Characterization of rBM-MSC To analyse the expressionof cell surface markers on rBM-MSCs cells at passage 3were subjected to the immunophenotyping Flow cytometryresult showed that rBM-MSCs are unequivocally positivefor CD901 (948) CD44H (416) CD29 (997) andCD71 (127) and negative for hematopoietic markers CD45(40) and CD11bc (43) as shown in Figure 2(a) To assessthe mesodermal differentiation ability of rBM-MSCs cellsat passage 3 were grown to the confluency and induced todifferentiate into adipocytes and osteocytes using relevantinduction media Following 20 days of adipogenic inductionlipid vacuoles were detected by positive staining of OilRed O whereas osteogenic differentiation was detected by


Day 0 Day 3

Day 5 Day 7

(a)

HDMEM

LDMEM

RPMI

DMEMF12

10 FBS 20 FBS

(b)

LDMEM HDMEM RPMI DMEMF12

Media

10 FBS

20 FBS

0

10

20

30

40

50

60

Nu

mb

er o

f co

lon

ies

lowastlowast

lowast

(c)


times105

10 FBS

20 FBS

lowastlowast

lowast

0

5

10

15

20

25

30

35

40

Nu

mb

er o

f ce

lls

(d)

Figure 1 Generation and optimization of rBM-MSCs culture Bone marrow was harvested from femur and tibia of SD rats and nucleatedcells were cultured in T25 flask in day 0 By day 3 cells began to attach and heterogeneous population with predominant fibroblast-likemorphology were observed by day 7 (a) One million of nucleated cells from bone marrow were cultured for 10 days in respective media andFBS concentrations Colonies were subjected to crystal violet staining and colonies which brightly stained were counted (b) Four differentbasal media with 10 and 20 FBS concentration were utilized to culture 1 times 106 freshly isolated BMnucleated cells for CFU and proliferationassays CFU-f and proliferation assays were measured using crystal violet staining and trypan blue exclusion test respectively Results wererepresentative of three independent experiments lowastSignificant values were compared between LDMEM 20 and other basal media with 20FBS significant values were compared between 10 FBS and 20 FBS of the respective media 119901 le 005 Microscopic magnification 200x

positive staining of Alizarin Red solution (Figure 2(b))Cell cultured in expansion media (negative control) showedneither detectable lipid vacuoles nor calcium depositionTo further confirm the mesodermal differentiation geneexpression analysis of adipocytes and osteocytes specific

genes was conducted using RT-PCRThe selected adipocytes(PPAR120574 and CEBPA) and osteocytes gene (osteopontinand osteonectin) were analysed Undifferentiated rBM-MSC(negative control) showed a faint expression of adipocytesand osteocytes genes Differentiated rBM-MSCs (osteocytes


250

200

150

100

50

SSC

-A(times

100

0)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)(times

100

0)

(times1

000

)

(times1

000

)

250

200

150

100

50

SSC

-A

(times1 000)

50 100 150 200 250

FSC-A

Population gate

102 103 104 105

FITC-A

Isotype control-FITC

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

Isotype control-PE

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD29

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD44H

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD71

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD45

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD901

250

200

150

100

50SS

C-A

102 103 104 105

PE-A

CD11bc

(a)

Control With induction

Adipocyte

Osteocyte

200120583m 200120583m

200120583m200120583m

(b)

Figure 2 Continued


L 1 2 3 4 5 6 7 8 9 10 11

360

GAPDH GAPDH GAPDH OP ON PP CE OP ON PP CEControl OS AD

OS osteocyte

AD adipocyte

(c)

Figure 2 Characterization of rBM-MSCs The immunophenotyping to characterize the surface markers was performed using rBM-MSCfrom passage 3 Cells were positively expressed as CD901 CD29 CD71 and CD44 and negatively expressed as CD11bc and CD45 (a)Mesodermal differentiationwas conducted using cells of passage 3 that is subjected to the relevant inductionmedia After 20 days of inductioncells were stained with Oil Red O solution and Alizarin Red solution respectively Adipogenic differentiation was evidence by lipid dropletformation stained with Oil Red O whereas osteogenic differentiation was evidenced by calcium deposits stained with Alizarin Red (b) Geneexpression of differentiated adipocytes and osteocytes was evaluated using RT-PCR Untreated rBM-MSC (control) showed faint expressionfor osteocytes (c) Results were representative of 3 experiments OP osteopontin ON osteonectin PP PPAR120574 and CE CEBPAMicroscopicmagnification 200x

and adipocytes) showed higher expression of adipocytesand osteocytes genes as compared to control as shown inFigure 2(c)

33 Growth Kinetics and Doubling Time of rBM-MSC Thegrowth pattern of rBM-MSCs was tracked by themorpholog-ical assessment growth kinetics curve and doubling time atvarious passagesThemorphology of adherent cells appearedto be relatively smaller and defined at passage 1 till passage 3(Figure 3(a)) It was noted that cells were assuming a uniformspindle shape till passage 3 however the morphology hasgradually changed from passage 4 onward into amore flattenlarger and polygonal phenotypes Although the growth curveof an individual cell passage from passages 1ndash5 had depictedan accepted sigmoid shape or exponential growth patternthe duration of lag log and plateau phases is varied amongnumbers of passages (Figure 3(b)) Passages 1ndash3 cells reflectedan initial lag phase for 1 day followedby exponential log phasefor 4 days and then a plateau phase It was observed thatpassage 1 showed an abrupt log phase while other passages(P2-3) showed a gradual increase of log phase However theproliferative stage of log phase is not being spotted in cellsfrom passages 4-5 which is an indication of growth arrestThe growth arrest of passages 4-5 rBM-MSCs was furtherconfirmed with deduced doubling time The doubling timeof passages 1ndash3 rBM-MSCs is within 20ndash30 hours whereaspassage 5 shows an extreme of 130 hours as shown inFigure 3(c)

34 Rat Bone Morrow MSCs Undergo Cellular Senescenceand Cell Cycle Arrest Since rBM-MSCs from passages 1ndash5exhibited various growth kinetic patterns with compromisedlog phases at late passages another mean of measurementwas opted to verify this phenomenon The proliferation rateof rBM-MSCs was further rectified using tritiated thymidine(3H-Tdr) incorporation assay The highest proliferation was

Table 4 Percentage of viability and early apoptosis at variouspassages of rBM-MSC

Passage Viable cells () Early apoptosis ()1 301 plusmn 10 684 plusmn 04

2 424 plusmn 30 568 plusmn 30




observed at 48 hours in all passages However the active pro-liferation was only noted at passages 1ndash3 while cell expansionwas halted at passages 4-5 (Figure 4(a)) The complete haltin cell proliferation at passages 4-5 was further decipheredby the occurrence of senescence When rBM-MSCs frompassage 2 and passage 5 were stained with senescencemarkerSA 120573-gal only passage 5 rBM-MSCs were significantlypositive for SA 120573-gal as evidence by blue precipitation withinthe cells while actively proliferating rBM-MSCs in passage 2showed negative staining for 120573-gal (pH 6) (Figure 4(b)) Tostudy the cell cycle pattern of rBM-MSC DNA content ofrBM-MSC at passage 2 and passage 5 was measured usingPI staining Result showed that almost all cells in passage 5were arrested at G0G1 phase (G0G1 = 9703 S = 019 andG2M = 278) as compared to passage 2 (G0G1 = 8585 S= 865 and G2M = 55) as shown in Figure 4(c)

35 Senescence of rBM-MSCs Is Not Associated with ApoptosisInduction Apoptosis assay was performed to assess the via-bility of rBM-MSCs at various passages (P1-P0) whether thedocumented cellular senescence is caused by the induction ofapoptosis The apoptosis results revealed that as the passagewas increased the percentage of early apoptosis decreasedwhile the percentage of viability is increased as shown inTable 4


Passage 0 Passage 1 Passage 2


(a)

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

Nu

mb

er o

f ce

lls

Day

Passage 1

Passage 2

Passage 3

Passage 4

Passage 5

times105

(b)

1 2 3 4 5

Passage

0

20

40

60

80

100

120

140

Do

ub

lin

g ti

me

(ho

ur)

(c)

Figure 3 Growth kinetics and doubling time of rBM-MSC Morphological observation of rBM-MSC cultured in LDMEM (20 FBS) in thepresence of 20 ngmL 1 ITS and 1 NEAA at various passages Cells were successfully expanded until passage 5 and assumed a spindle-shaped fibroblast-like morphology As the passage increased polygonal and flatten shaped cells were predominating (a) Rat BM-MSCs wereplated in 6-well plate at 150000 cellswell andmedia were changed every 2 days for 6 days Cells depicted an initial lag phase for 1 day followedby exponential log phase for 4 days and then a plateau phase was observed (b) Doubling time was determined by Patterson Formula (lowast) andexpressed as mean doubling time (c) The result represented the mean plusmn SD Microscopic magnification 200x

4 Discussion

Various studies have been conducted using animal modelsin attempt to evaluate the potential use of MSCs in clinicalapplications As preclinical study is vital for clinical trialsthis requires an establishment of animal-based MSCs culturesystem through a feasible isolation and expansion procedureat in vitro setting [15 46 47] Several methods have beenemployed to isolate MSCs mainly based on its plasticadherence property whereby whole bone marrow (BM) is

aspirated and directly plated into culture dish This is thestandard method of isolation that is mostly used in previousstudies due to the cost effectiveness straight forward andless laborious nature of the procedures [48] Besides plasticadherence methods of refining MSC population such asdensity gradient separation technique [42 49 50] and enrich-ment of MSCs culture using magnetic cell sorting [51] werealso tested widely Previous studies have suggested thatMSCsisolated from whole BM exhibited the superior cell growthin terms of absolute cell numbers negligible hematopoietic


0

1000

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7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

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500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

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1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)

Figure 4 Cellular senescence and cell cycle arrest of culture expanded rBM-MSCs Cells from passages 1ndash5 were seeded at 5000 cellswellcultured in 96-well plate for 24 h 48 h and 72 h Cultures were pulsed with 10 120583L 3H-TdR at 24 hours prior to measurement The rate ofproliferation decreased as the passage increased (a) Cells at passage 2 and passage 5 were fixed with 70 ethanol for 10 minutes and stainedwith X-gal in the respective buffer overnight Cells at passages 2 and 5 were stained positive with lysosomal 120573-gal (pH 4) staining showing thatcells were expressing lysosomal 120573-gal in early passage as well as in late passage Cells at passage 5 were stained positive for SA-120573-gal (pH 6)staining as evidenced by blue precipitation but not at passage 2 Cells at passage 2 and passage 5 were cultured in 25 cm2 flasks Upon reaching80ndash90 confluency cells were harvested and subjected to PI staining Cells were analysed using flow cytometer Results were representativeof three independent experiments Microscopic magnification 200x


contamination higher CFU-f number and a longer telomerelength as compared to Percoll and Ficoll density separationtechniques [50] Hence the present study has utilized a directplating method of culturing whole BM from femur and tibiaof Sprague Dawley rats

The initial primary culture (P0) of BM was mixed withother cell populations which did not allow a clear discrim-ination between MSCs and other adherent cells Howeveras the number of passages was increasing the adherent cellculture became more morphologically homogenous Alongwith MSCs BM cultures at passage 0 were also found to becomprised of other primary BM cells such as macrophagesand endothelial cells that promote heterogeneity of pri-mary culture Since the present study has utilized a wholeBM the contamination of other BM residing cells namelyhematopoietic cells fat cells endothelial cells and fibroblaststhatmay retain up to fewer passages of BMculture could con-tribute to formation of mixed populations [52] Nonethelessmix populations of early bonemarrow cells culture are crucialas the growth factors andmulticellular interaction with othercells are needed for the initial colony forming and expansionof MSCs [53]

To date there is no standard method for culturing rBM-MSCs It is difficult to compare methods of culturing MSCsas there are high inconsistency among different laboratoriessuch as choice ofmedia the type of serum plating density theaddition of supplements and level of confluency which playa crucial role in MSCs culture as it can affect the expansiondifferentiation and immunogenic properties of MSCs [50]We have employed colony forming unit-fibroblast (CFU-f)assay to optimize the culture condition for rBM-MSC asthis assay allows the identification of adherent cells withstem cell properties that expand to form colonies [42] Thecurrent findings demonstrated that LDMEM basal mediumwith 20 FBS served as the optimal condition for rBM-MSCsexpansion yielding the highest CFU-f count In linewith thisAyatollahi et al (2012) had reported the excellent tropic effectof LDMEMbasalmedium to support and enhance the growthofMSCs as compared to the120572MEMandHDMEMAmong alltested concentrations of FBS 15 of supplemented FBS hadshown a higher cell yield as compared to the lower concentra-tions namely 5 and 10 of FBS [54] Since expanding rBM-MSCs from the primary culture are considered an arduousprocess and thus a higher concentration of FBS 20 wasopted and compared to the conventional 10 of FBS Asexpected 20 of FBS level has induced a higher number ofcolonies in all media as compared to 10 FBS (Figure 1)However it is still unable to decipher whether the increasedconcentration of FBS has potentially slowed down the cellularsenescence although 20 FBS supplemented rBM-MSCswere expanded till passage 45 but the conventional FBSconcentration fails to propagate cell number at very earlypassages

High glucose DMEM and DMEMf12 are among com-monly used media for culturing adherent cells especiallyMSCs while RPMI are routinely used for culturing lympho-cytes [46ndash60] In this study we have also consumed RPMIbasal medium to culture rBM-MSC In fact rBM-MSCswere able to grow in RPMI although the CFU-f count was

lower than LDMEM Overall culture medium that containsHDMEM has contributed the lowest number of CFU-f ascompared to LDMEMDMEMf12 andRPMI Consumptionof the basal medium that contains a high glucose levelmay jeopardise the beneficiary effects of culturing stem cellsover a low glucose culture medium It has been shown thatculturing ratMSCs in high glucosemedium is associatedwithcellular senescence while low glucose medium is enhancingproliferation and CFU counts while reducing the apoptosis[61] Glucose is crucial source of cellular energy however theelevated concentration of glucose can raise the productionof reactive oxygen species and thus leads to cell damageand cellular senescence [62] Due to balanced oxidativestress in low glucose medium stem cells might be stress-freeand physiologically unchallenged since stem cells are verysensitive to the changes in the microenvironment

Although the colonies formed in LDMEM culturemedium are highest further cultivation in the same mediumwith 20 FBS did not support the expansion of rBM-MSCsThis phenomenon is also observed in other basal media andthus prompted us to use additional growth factors Basic FGFwas used to enhance the growth of rBM-MSCs in our studyas it is the most common growth factor that is known toinduce proliferation of MSCs [63ndash66] To further improverBM-MSC growth 1 ITS and 1 NEAA were also added inculture along with 20 ngmL bFGFThe addition of bFGF andsupplements significantly altered the cell size and appearanceof rBM-MSC as compared to rBM-MSC cultured withoutany supplements (data not shown) It is well establishedthat MSCs at in vitro culture contain two distinct types ofmorphologies (1) small spindle-shaped fibroblast-like cellswhich proliferate rapidly and (2) large flattened cells withslower proliferation rate [16 46 50 57] Similar to what hasbeen reported rBM-MSCs cultured in optimized mediumand supplements appeared as spindle-shaped and fibroblast-like but when the number of passages increased a gradualdomination of large and flattened cells was noted Similarobservations were also reported by previous studies wherebythe number of large and flat cells increased over the time incultures [49 57] This transition could be due to losing ofcell integrity and also loss of autocrine and paracrine effectsresulting in inadequate formation of microenvironment thatis necessary for MSCs proliferation Furthermore it couldalso be due to an autocommitment of MSCs towards maturemesodermal lineages Based on RT-PCR data it has beenshown that MSCs at resting condition without inductivemedia are slightly expressed RNAs that lead to adipogenicand osteogenic differentiation This could be accountable forthe slow growth rate of MSCs during the expansion periodwhere culture-driven cellular commitment towards maturecells reduces the self-renewal and propagation of stem cellpopulation

Mesenchymal stem cells characterization mainly relieson the assessment of (1) surface antigen markers expressionand (2) the ability of MSC to differentiate into mesodermallineages (osteocytes adipocytes and chondrocytes) To datethere are no specific markers for MSCs thus a combinationof commonly accepted positive and negative markers wasopted for the MSCs immunophenotyping We have shown


that rBM-MSCs were positive for CD29 CD901 CD44Hand CD71 and negative for hematopoietic markers CD45 andCD11bc [15 42 48 54 65] Upon induction rBM-MSCswere able to differentiate into adipocytes and osteocytes andthis finding was further confirmed by expression of osteo-cytes (osteopontin and osteonectin) and adipocytes (PPAR120574and CEBPA) genes Interestingly rBM-MSCs without theinduction medium also expressed a low level of these genesOthers have also reported similar findings where the positiveexpression of osteonectin and osteopontin and expressionof adipogenic gene (PPAR120574) were detected in MSCs in theabsence of induction medium [16 67]The expression of adi-pogenic and osteogenic genes in undifferentiated rBM-MSCsmight be the reason why the cells easily reach senescence andcan readily differentiate into mesenchymal stem cellsrsquo ownmesodermal lineages

To determine the proliferative capacity of rBM-MSCstritiated thymidine (3H-TdR) and growth kinetic assays wereperformed Rat BM-MSCs were spotted to proliferate rapidlyat passage 1 until passage 3 while reduction in proliferationwas evidenced at passage 3 onwards and almost ceased atpassage 5 These results were further confirmed with 3H-TdR assay and deduced doubling time Cells at passages 1ndash3 had maintained a stable doubling time (30 hrs) howeverthe doubling time was started to upraise at passage 4 (50 hrs)and reached maximum at passage 5 (130 hrs) However thecells were unable to be further expanded beyond passage 5due to the complete halt in cell growth despite the use ofoptimized culture medium This observation is not a newphenomenon as Liu et al (2003) also reported a similarfinding that rBM-MSCs from Wister rats were only beingcultured till 4 successful passages [49]

The ceased proliferation of passage 5 rBM-MSCs hastriggered the need for further exploration of the possibilityof apoptosis or senescence The apoptosis result was not inagreement with the spotted growth arrest of passage 4-5cells Surprisingly the viability of cells was improved with thenumber of passages However a substantial fraction of cells atall passages was undergoing early apoptosis process but notlate apoptosis as shown in Table 4 The expression of higherearly apoptosis in P1ndash3 cells was accompanied by neitherprogression towards late apoptosis nor cell death However itcould be possible that those cells in early apoptosis are able torecover as the translocation of PS was reversible in apoptosisinduced by tumour suppressor protein p53 provided theapoptosis stimuli were removed [68]

Our data also revealed that rBM-MSCs almost cease toproliferate at passage 5 and morphology of cells was largeand flattened which are characteristics of cellular senescence120573-gal is found in lysosomes of all types of cells at all stagesincluding proliferating quiescent and senescence cells andcan be detected by X-gal staining at pH 4 [44] MeanwhileSA120573-gal staining is detectable at pH60 and expressed by cellsthat undergo senescence but not in actively proliferating cells[44 69] Result of X-gal staining on passage 2 and passage5 rBM-MSC showed a positive staining for senescence inpassage 5 rBM-MSCs but not in passage 2 Lysosomal 120573-gal assay was also performed on rBM-MSC in passage 2and passage 5 and our finding showed positive staining for

lysosomal 120573-gal However since MSCs almost ceased inproliferation as indicated in Figure 3 we were unable tofurther passage the MSCs and explore the senescence in laterpassages

The hallmark of senescence is the inability of cells toadvance into cell cycle Senescence cells show growth arrestat G1 phase of cell cycle despite sufficient growth conditioncells still fail to initiate DNA replication [70] Cells at passage5 were mainly arrested at G0G1 phase of cell cycle On theother hand rBM-MSCs at passage 2 stained which is negativefor SA 120573-gal showing a progression in cell cycle with higherpercentage of S and G2M phase as compared to passage5 The reduced proliferation changes in morphology fromspindle-shape fibroblast-like cells into large flattened cellspositive staining for SA120573-gal and growth arrest of rBM-MSCpassage 5 at G0G1 phase of cell cycle could be an evidence ofcells undergoing senescence

Study on human fibroblasts demonstrated that underan inadequate culture condition (025 FBS) the cellsunderwent a premature growth arrest When fibroblasts wereinitially expanded in 10 FBS and later grown in 025FBS they showed a substantial arrest in cellular growthHowever the cell growth recovered and cell cycle machinerywas activated when the culture was reconstituted with 10FBS In fact transfection of HTERT (human telomerasereverse transcriptase) as well did not abolish the growthretardation in the presence of 025 of FBS [71] Regardlessof the elongation of telomerase length serum deprivationelevated p16INK4a a tumour suppressor protein that inhibitscyclin-dependent kinases (CDKs) and leads to a permanentgrowth arrest [72] Similarly human MSCs that underwent apremature growth arrest during the in vitro culture as wellshowed a significant change in p16INK4a level [73] It waselegantly shown that the suppression of p16INK4a in humanMSCs by wild-type p53 inducible phosphatase-1 (Wip-1) astress modulator extended the life-span of human MSCswhile maintaining its multipotent differentiation potential[73]

In conclusion the present study has successfully gener-ated and characterized the rBM-MSCs from SD rats withoptimized culture conditions based on the choice of basalmedium concentration of FBS and growth factor supple-ments The basal media LDMEM and 20 FBS provide theoptimal culture condition for expanding rBM-MSC and theaddition of bFGF ITS and NEAA significantly enhancedthe morphology and proliferation capacity of rBM-MSCs atvery early passages Under the in vitro culture conditioneven with optimized culture conditions rBM-MSCs wereundergoing cellular senescence which may relate to thegradual autocommitment of rBM-MSC into mature cellsThus further research is necessary to understand the internaland external cues that trigger the process of senescence inculture expanded rBM-MSCs

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper and regarding thefunding that they have received


Acknowledgments

This research work is supported by Fundamental ResearchGrant Scheme (FRGS) 04-01-12-1131FR and Research Uni-versity Grant Scheme (RUGS) 04-02-12-1757RU

References

[1] A J Friedenstein R K Chailakhjan and K S Lalykina ldquoThedevelopment of fibroblast colonies in monolayer cultures ofguinea-pig bonemarrow and spleen cellsrdquoCell Proliferation vol3 no 4 pp 393ndash403 1970

[2] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[3] M C Stewart and A A Stewart ldquoMesenchymal stem cellscharacteristics sources and mechanisms of actionrdquo VeterinaryClinics of North America Equine Practice vol 27 no 2 pp 243ndash261 2011

[4] C-D Wan R Cheng H-B Wang and T Liu ldquoImmunomod-ulatory effects of mesenchymal stem cells derived from adiposetissues in a rat orthotopic liver transplantation modelrdquo Hepa-tobiliary and Pancreatic Diseases International vol 7 no 1 pp29ndash33 2008

[5] M Giuliani M Fleury A Vernochet et al ldquoLong-lastinginhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferationrdquo PLoS ONE vol 6 no 5 Article IDe19988 2011

[6] H E Young T A Steele R A Bray et al ldquoHuman reservepluripotent mesenchymal stem cells are present in the connec-tive tissues of skeletal muscle and dermis derived from fetaladult and geriatric donorsrdquo The Anatomical Record vol 264no 1 pp 51ndash62 2001

[7] L-L Lu Y-J Liu S-G Yang et al ldquoIsolation and charac-terization of human umbilical cord mesenchymal stem cellswith hematopoiesis-supportive function and other potentialsrdquoHaematologica vol 91 no 8 pp 1017ndash1026 2006

[8] O K Lee T K Kuo W-M Chen K-D Lee S-L Hsieh andT-H Chen ldquoIsolation of multipotent mesenchymal stem cellsfrom umbilical cord bloodrdquo Blood vol 103 no 5 pp 1669ndash16752004

[9] T C Tran K Kimura M Nagano et al ldquoIdentification ofhuman placenta-derived mesenchymal stem cells involved inre-endothelializationrdquo Journal of Cellular Physiology vol 226no 1 pp 224ndash235 2011

[10] A Nimura T Muneta H Koga et al ldquoIncreased proliferationof human synovial mesenchymal stem cells with autologoushuman serum comparisons with bone marrow mesenchymalstem cells and with fetal bovine serumrdquo Arthritis and Rheuma-tism vol 58 no 2 pp 501ndash510 2008

[11] K Bieback S Kern H Kluter andH Eichler ldquoCritical parame-ters for the isolation of mesenchymal stem cells from umbilicalcord bloodrdquo STEM CELLS vol 22 no 4 pp 625ndash634 2004

[12] A Peister J A Mellad B L Larson B M Hall L F Gibsonand D J Prockop ldquoAdult stem cells from bone marrow (MSCs)isolated from different strains of inbred mice vary in surfaceepitopes rates of proliferation and differentiation potentialrdquoBlood vol 103 no 5 pp 1662ndash1668 2004

[13] G Ren J Su L Zhang et al ldquoSpecies variation in themechanisms of mesenchymal stem cell-mediated immunosup-pressionrdquo Stem Cells vol 27 no 8 pp 1954ndash1962 2009

[14] S M Devine A M Bartholomew N Mahmud et al ldquoMes-enchymal stem cells are capable of homing to the bone marrowof non-human primates following systemic infusionrdquo Experi-mental Hematology vol 29 no 2 pp 244ndash255 2001

[15] E H Javazon D C Colter E J Schwarz and D J ProckopldquoRat marrow stromal cells are more sensitive to plating densityand expandmore rapidly from single-cell-derived colonies thanhuman marrow stromal cellsrdquo STEM CELLS vol 19 no 3 pp219ndash225 2001

[16] E Karaoz A Aksoy S Ayhan A E SarIboyacI F Kaymaz andM Kasap ldquoCharacterization of mesenchymal stem cells fromrat bone marrow ultrastructural properties differentiationpotential and immunophenotypicmarkersrdquoHistochemistry andCell Biology vol 132 no 5 pp 533ndash546 2009

[17] B Johnstone T M Hering A I Caplan V M Goldberg andJ U Yoo ldquoIn vitro chondrogenesis of bone marrow-derivedmesenchymal progenitor cellsrdquo Experimental Cell Research vol238 no 1 pp 265ndash272 1998

[18] M B Eslaminejad S Mardpour andM Ebrahimi ldquoMesenchy-mal stem cells derived from rat epicardial versus epididymaladipose tissuerdquo Iranian Journal of Basic Medical Sciences vol14 no 1 pp 25ndash34 2011

[19] D R Martin N R Cox T L Hathcock G P Niemeyer and HJ Baker ldquoIsolation and characterization of multipotential mes-enchymal stem cells from feline bone marrowrdquo ExperimentalHematology vol 30 no 8 pp 879ndash886 2002

[20] S Violini P Ramelli L F Pisani C Gorni and P MarianildquoHorse bone marrow mesenchymal stem cells express embryostem cell markers and show the ability for tenogenic differenti-ation by in vitro exposure to BMP-12rdquo BMCCell Biology vol 10article 29 2009

[21] Q-P Xie H Huang B Xu et al ldquoHuman bone marrowmesenchymal stem cells differentiate into insulin-producingcells upon microenvironmental manipulation in vitrordquo Differ-entiation vol 77 no 5 pp 483ndash491 2009

[22] S H Hong E J Gang J A Jeong et al ldquoIn vitro differentiationof human umbilical cord blood-derived mesenchymal stemcells into hepatocyte-like cellsrdquo Biochemical and BiophysicalResearch Communications vol 330 no 4 pp 1153ndash1161 2005

[23] W Xu X Zhang H Qian et al ldquoMesenchymal stem cells fromadult human bone marrow differentiate into a cardiomyocytephenotype in vitrordquo Experimental Biology and Medicine vol229 no 7 pp 623ndash631 2004

[24] E J Gang J A Jeong S H Hong et al ldquoSkeletal myogenicdifferentiation of mesenchymal stem cells isolated from humanumbilical cord bloodrdquo STEMCELLS vol 22 no 4 pp 617ndash6242004

[25] L Hou H Cao D Wang et al ldquoInduction of umbilical cordblood mesenchymal stem cells into neuron-like cells in vitrordquoInternational Journal of Hematology vol 78 no 3 pp 256ndash2612003

[26] J Oswald S Boxberger B Joslashrgensen et al ldquoMesenchymal stemcells can be differentiated into endothelial cells in vitrordquo STEMCELLS vol 22 no 3 pp 377ndash384 2004

[27] J-H Kim Y-S Kim K Park et al ldquoAntitumor efficacy ofcisplatin-loaded glycol chitosan nanoparticles in tumor-bearingmicerdquo Journal of Controlled Release vol 127 no 1 pp 41ndash492008

[28] K Guo J P Tang L Jie et al ldquoEngineering the first chimericantibody in targeting intracellular PRL-3 oncoprotein for can-cer therapy in micerdquo Oncotarget vol 3 no 2 pp 158ndash171 2012


[29] S D Kobayashi N Malachowa A R Whitney et al ldquoCom-parative analysis of USA300 virulence determinants in a rabbitmodel of skin and soft tissue infectionrdquo Journal of InfectiousDiseases vol 204 no 6 pp 937ndash941 2011

[30] S J Ferng D E Gonzalez M N Nguyen S J Sherman T Falkand H L Rilo ldquoEvaluation of a Parkinsonrsquos disease model inmedaka fishrdquo The FASEB Journal vol 27 no 1 supplement p5671 2013

[31] N Tajiri T Yasuhara T Shingo et al ldquoExercise exerts neuro-protective effects on Parkinsonrsquos disease model of ratsrdquo BrainResearch vol 1310 pp 200ndash207 2010

[32] L Fairbairn R Kapetanovic D P Sester and D A HumeldquoThe mononuclear phagocyte system of the pig as a model forunderstanding human innate immunity and diseaserdquo Journal ofLeukocyte Biology vol 89 no 6 pp 855ndash871 2011

[33] J Kocerha Yuhong Liu Kate Nelson et al ldquoNoncoding RNAdysregulation in a longitudinal study of peripheral blood from atransgenic Huntingtonrsquos disease monkey modelrdquo in Proceedingsof the Society for Neuroscience Conference 2012

[34] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[35] Z Qu-Petersen B Deasy R Jankowski et al ldquoIdentification ofa novel population of muscle stem cells in mice potential formuscle regenerationrdquoThe Journal of Cell Biology vol 157 no 5pp 851ndash864 2002

[36] D J Simmons P Seitz L Kidder et al ldquoPartial characterizationof rat marrow stromal cellsrdquo Calcified Tissue International vol48 no 5 pp 326ndash334 1991

[37] J E Aubin ldquoOsteoprogenitor cell frequency in rat bonemarrowstromal populations role for heterotypic cell-cell interactions inosteoblast differentiationrdquo Journal of Cellular Biochemistry vol72 no 3 pp 396ndash410 1999

[38] L K Chelluri R Kancherla N Turlapati et al ldquoImproveddifferentiation protocol of rat bone marrow precursors tofunctional islet like cellsrdquo Stem Cell Studies vol 1 no 1 articlee5 2011

[39] Z Neshati MMMatin A R Bahrami and AMoghimi ldquoDif-ferentiation of mesenchymal stem cells to insulin-producingcells and their impact on type 1 diabetic ratsrdquo Journal ofPhysiology and Biochemistry vol 66 no 2 pp 181ndash187 2010

[40] M Sgodda H Aurich S Kleist et al ldquoHepatocyte differenti-ation of mesenchymal stem cells from rat peritoneal adiposetissue in vitro and in vivordquo Experimental Cell Research vol 313no 13 pp 2875ndash2886 2007

[41] Y-C Shang S-H Wang F Xiong et al ldquoWnt3a signaling pro-motes proliferation myogenic differentiation and migration ofrat bonemarrowmesenchymal stemcellsrdquoActa PharmacologicaSinica vol 28 no 11 pp 1761ndash1774 2007

[42] H Yoshimura T Muneta A Nimura A Yokoyama H Kogaand I Sekiya ldquoComparison of rat mesenchymal stem cellsderived from bone marrow synovium periosteum adiposetissue and musclerdquo Cell and Tissue Research vol 327 no 3 pp449ndash462 2007

[43] D J Kurz S Decary Y Hong and J D ErusalimskyldquoSenescence-associated 120573-galactosidase reflects an increase inlysosomal mass during replicative ageing of human endothelialcellsrdquo Journal of Cell Science vol 113 no 20 pp 3613ndash36222000

[44] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[45] J P Horwitz J Chua R J Curby et al ldquoSubstrates forcytochemical demonstration of enzyme activity I Some substi-tuted 3-indolyl-I2-D-glycopyranosides1ardquo Journal of MedicinalChemistry vol 7 no 4 pp 574ndash575 1964

[46] D C Colter I Sekiya and D J Prockop ldquoIdentification ofa subpopulation of rapidly self-renewing and multipotentialadult stem cells in colonies of human marrow stromal cellsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 14 pp 7841ndash7845 2001

[47] G Fan L Wen M Li et al ldquoIsolation of mouse mesenchymalstem cells with normal ploidy from bone marrows by reducingoxidative stress in combination with extracellular matrixrdquo BMCCell Biology vol 12 no 1 article 30 2011

[48] E Karaoz A Okcu G Gacar O Saglam S Yuruker and HKenar ldquoA comprehensive characterization study of human bonemarrow mscs with an emphasis on molecular and ultrastruc-tural propertiesrdquo Journal of Cellular Physiology vol 226 no 5pp 1367ndash1382 2011

[49] Y Liu J Song W Liu Y Wan X Chen and C Hu ldquoGrowthand differentiation of rat bone marrow stromal cells does5-azacytidine trigger their cardiomyogenic differentiationrdquoCardiovascular Research vol 58 no 2 pp 460ndash468 2003

[50] K Mareschi D Rustichelli R Calabrese et al ldquoMultipotentmesenchymal stromal stem cell expansion by plating wholebone marrow at a low cellular density a more advantageousmethod for clinical userdquo Stem Cells International vol 2012Article ID 920581 10 pages 2012

[51] L Zhang and C Chan ldquoIsolation and enrichment of ratmesenchymal stem cells (MSCs) and separation of single-colony derived MSCsrdquo Journal of Visualized Experiments no37 2010

[52] H Castro-Malaspina R E Gay G Resnick et al ldquoCharacteri-zation of human bone marrow fibroblast colony-forming cells(CFU-F) and their progenyrdquo Blood vol 56 no 2 pp 289ndash3011980

[53] S Nadri M Soleimani R H Hosseni M Massumi A Atashiand R Izadpanah ldquoAn efficient method for isolation of murinebone marrow mesenchymal stem cellsrdquo International Journal ofDevelopmental Biology vol 51 no 8 pp 723ndash729 2007

[54] M Ayatollahi M K Salmani B Geramizadeh S Z Tabei MSoleimani andMH Sanati ldquoConditions to improve expansionof humanmesenchymal stem cells based on rat samplesrdquoWorldJournal of Stem Cells vol 4 no 1 pp 1ndash8 2012

[55] A Bartholomew C Sturgeon M Siatskas et al ldquoMesenchymalstem cells suppress lymphocyte proliferation in vitro andprolong skin graft survival in vivordquo Experimental Hematologyvol 30 no 1 pp 42ndash48 2002

[56] TDeuseM StubbendorffK Tang-Quan et al ldquoImmunogenic-ity and immunomodulatory properties of umbilical cord liningmesenchymal stem cellsrdquoCell Transplantation vol 20 no 5 pp655ndash667 2011

[57] B Neuhuber S A Swanger L Howard A Mackay and IFischer ldquoEffects of plating density and culture time on bonemarrow stromal cell characteristicsrdquo Experimental Hematologyvol 36 no 9 pp 1176ndash1185 2008

[58] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCharacterization of the optimal culture


conditions for clinical scale production of humanmesenchymalstem cellsrdquo Stem Cells vol 24 no 2 pp 462ndash471 2006

[59] R Ramasamy C K TongWK Yip S Vellasamy B C Tan andH F Seow ldquoBasic fibroblast growth factor modulates cell cycleof human umbilical cord-derivedmesenchymal stem cellsrdquo CellProliferation vol 45 no 2 pp 132ndash139 2012

[60] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003

[61] A Stolzing N Coleman and A Scutt ldquoGlucose-inducedreplicative senescence inmesenchymal stemcellsrdquoRejuvenationResearch vol 9 no 1 pp 31ndash35 2006

[62] T-C Chang M-F Hsu and K K Wu ldquoHigh glucose stressinduces mesenchymal stromal cell senescence through up-regulating autophagy (LB842)rdquoThe FASEB Journal vol 28 no1 supplement LB842 2014

[63] S Tsutsumi A Shimazu K Miyazaki et al ldquoRetention of mul-tilineage differentiation potential of mesenchymal cells duringproliferation in response to FGFrdquo Biochemical and BiophysicalResearch Communications vol 288 no 2 pp 413ndash419 2001

[64] KHanada J E Dennis andA I Caplan ldquoStimulatory effects ofbasic fibroblast growth factor and bonemorphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derivedmesenchymal stem cellsrdquo Journal of Bone andMineral Researchvol 12 no 10 pp 1606ndash1614 1997

[65] U Nekanti L Mohanty P Venugopal S Balasubramanian STotey andMTa ldquoOptimization and scale-up ofWhartonrsquos jelly-derived mesenchymal stem cells for clinical applicationsrdquo StemCell Research vol 5 no 3 pp 244ndash254 2010

[66] L-E Zaragosi G Ailhaud and C Dani ldquoAutocrine fibroblastgrowth factor 2 signaling is critical for self-renewal of humanmultipotent adipose-derived stem cellsrdquo Stem Cells vol 24 no11 pp 2412ndash2419 2006

[67] L Peng Z Jia X Yin et al ldquoComparative analysis of mesenchy-mal stem cells from bonemarrow cartilage and adipose tissuerdquoStem Cells and Development vol 17 no 4 pp 761ndash773 2008

[68] F J Geske R Lieberman R Strange and L E GerschensonldquoEarly stages of p53-induced apoptosis are reversiblerdquo CellDeath and Differentiation vol 8 no 2 pp 182ndash191 2001

[69] B Van Der Loo M J Fenton and J D Erusalimsky ldquoCyto-chemical detection of a senescence-associated 120573-galactosidasein endothelial and smooth muscle cells from human and rabbitblood vesselsrdquo Experimental Cell Research vol 241 no 2 pp309ndash315 1998

[70] J Campisi and F DrsquoAdda Di Fagagna ldquoCellular senescencewhen bad things happen to good cellsrdquo Nature Reviews Molec-ular Cell Biology vol 8 no 9 pp 729ndash740 2007

[71] R D Ramirez C P Morales B-S Herbert et al ldquoPutativetelomere-independent mechanisms of replicative aging reflectinadequate growth conditionsrdquoGenes and Development vol 15no 4 pp 398ndash403 2001

[72] M Serrano A W Lin M E McCurrach D Beach and SW Lowe ldquoOncogenic ras provokes premature cell senescenceassociated with accumulation of p53 and p16INK4ardquo Cell vol 88no 5 pp 593ndash602 1997

[73] J-S Lee M-O Lee B-H Moon S H Shim A J Fornace Jrand H-J Cha ldquoSenescent growth arrest in mesenchymal stemcells is bypassed by Wip1-mediated downregulation of intrinsicstress signaling pathwaysrdquo STEMCELLS vol 27 no 8 pp 1963ndash1975 2009

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Table 1 Basal media and FBS concentration used for CFU-f assay

Basal media FBS concentration ()Roswell Park Memorial Institute (RPMI) with GLUTAMAX-I 10RPMI with GLUTAMAX-I 20Dulbeccorsquos Modified Eaglersquos Medium with nutrient mixture F12 (HAM) [1 1] DMEMF12) with GLUTAMAX-I 10DMEMF12 with GLUTAMAX-I 20Low glucose Dulbeccorsquos Modified Eaglersquos Medium (LDMEM) with GLUTAMAX-I 10LDMEM with GLUTAMAX-I 20High glucose Dulbeccorsquos Modified Eagle Medium (HDMEM) with GLUTAMAX-I 10HDMEM with GLUTAMAX-I 20

MSCs have also been isolated from several animal speciesnamely mouse [11ndash13] baboon [14] rat [15 16] monkey[13] rabbit [17] dog [18] cat [19] and horse [20] Despitethe initial understanding on targetedlimited differentiationtowardsmesodermal lineages the current studies have shownthat under a right stimulation MSCs are able to differentiateinto cells of endodermal and ectodermal origins such as 120573-pancreatic cells [21] hepatocyte [22] cardiomyocyte [23]skeletal muscle [24] neuron cells [25] and epithelial cells[26]

Many types of research that test the in vivo nature orfunctions of MSCs are still remarkably relying on animalmodels Employing animals in biological science has becomeindispensable as most of the pharmacological and toxico-logical studies have been developed using laboratory-basedanimals as testing tools For instance the convenience ofmicemodel with a possible genetic modification and availabilityof research reagents and species-specific antibodies ease thepreclinical studies [27 28] Beside mouse model some largerscale studies and disease-specificmodels were also developedusing another type of animal such as rat rabbit fish pigand monkey [29ndash33] In regard to current MSC researchhuman and mouse MSCs are extensively used in the presentresearch scenario as these MSCs are relatively easy to beharvested and expanded at in vitro culture [34 35] Whenconcerning other species it has been reported that the ratMSCs are difficult to isolate and culture-expanded at the invitro culture [15 36 37] This conundrum drives the stemcells research using rat MSCs which is not as lucrative andwell embraced as mouse counterpart However there is aneed for the rat-based MSCs especially in diabetic researchwhere most of the animal model investigations are tailoredbased on rats [38 39] One of the major concerns whicharise in rat MSCs culture is to maintain a stable in vitroculture that provides an uninterrupted supply of stem cellsfor ongoing laboratory and animalworksNumerous researchstudies have reported the paucities of rBM-MSC culturewhere after certain rounds of passages the cells were failed tobe amplified at adequate numbers Despite this observationthe exact mechanism of such growth retardation is not fullyelucidated Thus this research project is aimed at optimizingthe laboratory protocol for isolation characterization andexpansion of rat MSC to fill the gap in the current rat-basedMSCs studies

2 Materials and Methods

21 Animals Sprague Dawley (SD) rats (250ndash350 g and 5ndash8 weeks) were obtained from Chenur Supplier (KajangSelangor Malaysia) Animals were acclimatized and handledwith standard animal care procedures as prescribed byInstitutional Animal Care and Use Committee UniversitiPutra Malaysia (IACUC UPM) Animals were sacrificed bycervical dislocation

22 Generation of Rat Bone Marrow MSCs Bone marrowcells were obtained from Sprague Dawley (SD) rats byflushing femurs and tibiae and cultured in 25 cm2 flask in acomplete culture medium with 1 penicillinstreptomycin05 Fungizone and 01 gentamycin (Gibco United King-dom) After 72 hours of plating nonadherent cells wereremoved and medium was replaced at every 48 hoursAdherent cells were further propagated and upon reaching80ndash90 confluency cells were trypsinised by using 005trypsin-EDTA (Gibco United Kingdom) at 37∘C for 3ndash5minutes Harvested cells were cultured in 25 cm2 flasks forfurther expansion During expansion period media werechanged every 2 days Expanded cells were either used fordownstream experiments or cryopreserved using freezingmedia (10 DMSO and 90 FBS) Media used in rBM-MSC expansion were LDMEM with GLUTAMAX-I (GibcoUnited Kingdom) supplemented with 20 foetal bovineserum (FBS) 1 penicillinstreptomycin 05 Fungizoneand 01 gentamycin (GibcoUnitedKingdom) Supplementsused in the optimization were 20 ngmL basic fibroblastgrowth factor (bFGF) (RampD System USA) 1 nonessentialamino acids (NEAA) (Gibco United Kingdom) and 1insulin transferrin sodium selenite (ITS) (Sigma AldrichUSA)

23 Colony Forming Unit-Fibroblast Assay Colony formingunit-fibroblast (CFU-f) assay was conducted with 1 times 106of nucleated cells from freshly isolated rat bone marrowand seeded in 60mm2 cell culture dish (Becton DickinsonUSA) for 10 days Various basal media were consumed toassess CFU-f as presented in Table 1 and purchased fromGibco United Kingdom Complete media were constitutedwith an individual basalmedium supplementedwith 1peni-cillinstreptomycin 05 Fungizone and 01 gentamycin


























119879119889 =119879 log 2

log (1198731199051198730) (lowast)






3 Results




Day 0 Day 3

Day 5 Day 7

(a)

HDMEM

LDMEM

RPMI

DMEMF12

10 FBS 20 FBS

(b)


Media

10 FBS

20 FBS

0

10

20

30

40

50

60

Nu

mb

er o

f co

lon

ies

lowastlowast

lowast

(c)


times105

10 FBS

20 FBS

lowastlowast

lowast

0

5

10

15

20

25

30

35

40

Nu

mb

er o

f ce

lls

(d)





250

200

150

100

50

SSC

-A(times

100

0)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)(times

100

0)

(times1

000

)

(times1

000

)

250

200

150

100

50

SSC

-A

(times1 000)

50 100 150 200 250

FSC-A

Population gate

102 103 104 105

FITC-A


250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

Isotype control-PE

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD29

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD44H

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD71

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD45

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD901

250

200

150

100

50SS

C-A

102 103 104 105

PE-A

CD11bc

(a)


Adipocyte

Osteocyte

200120583m 200120583m

200120583m200120583m

(b)

Figure 2 Continued


L 1 2 3 4 5 6 7 8 9 10 11

360


OS osteocyte

AD adipocyte

(c)
















(a)

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

Nu

mb

er o

f ce

lls

Day

Passage 1

Passage 2

Passage 3

Passage 4

Passage 5

times105

(b)

1 2 3 4 5

Passage

0

20

40

60

80

100

120

140

Do

ub

lin

g ti

me

(ho

ur)

(c)


4 Discussion




0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

0

100

200

300

400

500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

300

600

900

1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)



















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























119879119889 =119879 log 2

log (1198731199051198730) (lowast)






3 Results




Day 0 Day 3

Day 5 Day 7

(a)

HDMEM

LDMEM

RPMI

DMEMF12

10 FBS 20 FBS

(b)


Media

10 FBS

20 FBS

0

10

20

30

40

50

60

Nu

mb

er o

f co

lon

ies

lowastlowast

lowast

(c)


times105

10 FBS

20 FBS

lowastlowast

lowast

0

5

10

15

20

25

30

35

40

Nu

mb

er o

f ce

lls

(d)





250

200

150

100

50

SSC

-A(times

100

0)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)(times

100

0)

(times1

000

)

(times1

000

)

250

200

150

100

50

SSC

-A

(times1 000)

50 100 150 200 250

FSC-A

Population gate

102 103 104 105

FITC-A


250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

Isotype control-PE

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD29

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD44H

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD71

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD45

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD901

250

200

150

100

50SS

C-A

102 103 104 105

PE-A

CD11bc

(a)


Adipocyte

Osteocyte

200120583m 200120583m

200120583m200120583m

(b)

Figure 2 Continued


L 1 2 3 4 5 6 7 8 9 10 11

360


OS osteocyte

AD adipocyte

(c)
















(a)

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

Nu

mb

er o

f ce

lls

Day

Passage 1

Passage 2

Passage 3

Passage 4

Passage 5

times105

(b)

1 2 3 4 5

Passage

0

20

40

60

80

100

120

140

Do

ub

lin

g ti

me

(ho

ur)

(c)


4 Discussion




0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

0

100

200

300

400

500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

300

600

900

1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)



















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Day 0 Day 3

Day 5 Day 7

(a)

HDMEM

LDMEM

RPMI

DMEMF12

10 FBS 20 FBS

(b)


Media

10 FBS

20 FBS

0

10

20

30

40

50

60

Nu

mb

er o

f co

lon

ies

lowastlowast

lowast

(c)


times105

10 FBS

20 FBS

lowastlowast

lowast

0

5

10

15

20

25

30

35

40

Nu

mb

er o

f ce

lls

(d)





250

200

150

100

50

SSC

-A(times

100

0)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)(times

100

0)

(times1

000

)

(times1

000

)

250

200

150

100

50

SSC

-A

(times1 000)

50 100 150 200 250

FSC-A

Population gate

102 103 104 105

FITC-A


250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

Isotype control-PE

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD29

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD44H

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD71

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD45

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD901

250

200

150

100

50SS

C-A

102 103 104 105

PE-A

CD11bc

(a)


Adipocyte

Osteocyte

200120583m 200120583m

200120583m200120583m

(b)

Figure 2 Continued


L 1 2 3 4 5 6 7 8 9 10 11

360


OS osteocyte

AD adipocyte

(c)
















(a)

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

Nu

mb

er o

f ce

lls

Day

Passage 1

Passage 2

Passage 3

Passage 4

Passage 5

times105

(b)

1 2 3 4 5

Passage

0

20

40

60

80

100

120

140

Do

ub

lin

g ti

me

(ho

ur)

(c)


4 Discussion




0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

0

100

200

300

400

500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

300

600

900

1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)



















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


250

200

150

100

50

SSC

-A(times

100

0)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)

(times1

000

)(times

100

0)

(times1

000

)

(times1

000

)

250

200

150

100

50

SSC

-A

(times1 000)

50 100 150 200 250

FSC-A

Population gate

102 103 104 105

FITC-A


250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

Isotype control-PE

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD29

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD44H

250

200

150

100

50

SSC

-A

102 103 104 105

FITC-A

CD71

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD45

250

200

150

100

50

SSC

-A

102 103 104 105

PE-A

CD901

250

200

150

100

50SS

C-A

102 103 104 105

PE-A

CD11bc

(a)


Adipocyte

Osteocyte

200120583m 200120583m

200120583m200120583m

(b)

Figure 2 Continued


L 1 2 3 4 5 6 7 8 9 10 11

360


OS osteocyte

AD adipocyte

(c)
















(a)

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

Nu

mb

er o

f ce

lls

Day

Passage 1

Passage 2

Passage 3

Passage 4

Passage 5

times105

(b)

1 2 3 4 5

Passage

0

20

40

60

80

100

120

140

Do

ub

lin

g ti

me

(ho

ur)

(c)


4 Discussion




0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

0

100

200

300

400

500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

300

600

900

1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)



















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


L 1 2 3 4 5 6 7 8 9 10 11

360


OS osteocyte

AD adipocyte

(c)
















(a)

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

Nu

mb

er o

f ce

lls

Day

Passage 1

Passage 2

Passage 3

Passage 4

Passage 5

times105

(b)

1 2 3 4 5

Passage

0

20

40

60

80

100

120

140

Do

ub

lin

g ti

me

(ho

ur)

(c)


4 Discussion




0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

0

100

200

300

400

500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

300

600

900

1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)



















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




(a)

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

Nu

mb

er o

f ce

lls

Day

Passage 1

Passage 2

Passage 3

Passage 4

Passage 5

times105

(b)

1 2 3 4 5

Passage

0

20

40

60

80

100

120

140

Do

ub

lin

g ti

me

(ho

ur)

(c)


4 Discussion




0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

0

100

200

300

400

500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

300

600

900

1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)



















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5C

ou

nt

per

min

ute

(C

PM

)Passage

24

48

72

(a)

Passage 2

Passage 5

pH 4 pH 6

200120583m 200120583m

200120583m200120583m

(b)

0 50 100 150 200

Channels (FL2-A)

0

100

200

300

400

500

Nu

mb

er

40 80 120 1600

Channels (FL2-A)

0

300

600

900

1200

Nu

mb

er

G0G1 = 859

S = 87

G2M = 55

G0G1 = 970

S = 02

G2M = 28

(c)



















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Competing Interests



Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article characterization and expression of

Documents