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Improving the expansion and neuronal differentiation of mesenchymal stem cells through culture

*Corresponding author.Tel.:+1-203-432-4262;fax:+1-203-432-0030.

E-mail address:mark.saltzman@https://www.wendangku.net/doc/5113270041.html,(W.M.Saltzman).

surface was coated with a varietyof substrates that have been widelyused in mesenchy mal and neural cultures, including poly-d-lysine,poly-l-lysine,collagen,laminin,?bronectin,and a soluble basement membrane extract known as Matrigel.These modi?ed surfaces were compared with standard tissue grade polystyrene for their effects on the growth and neuronal differentiation of MSCs.The effect of coating densitywas also examined.

2.Materials and methods

2.1.Materials

Frozen human MSCs and Trypsin/EDTA solution were purchased from BioWhittaker(Walkersville,MD). All culture media components,including a Minimum Essential Medium(a-MEM),Dulbecco’s Modi?ed Eagle Media(DMEM),fetal bovine serum(FBS),L-glutamate,basic Fibroblast Growth Factor(bFGF), penicillin,streptomycin,as well as Dulbecco’s phos-phate-buffered saline(D-PBS),were purchased from Gibco BRL(Rockville,MD).Collagen type I(bovine dermal)was obtained from Cohesion Technologies (Palo Alto,CA).The rest of the substrates,including poly-d-lysine,poly-l-lysine,laminin(mouse),?bronec-tin(human),and Matrigel(mouse)were from BD Biosciences(Bedford,MA).All chemicals,including dimethylsulfoxide(DMSO),butylated hydoxyanisole (BHA),potassium chloride(KCl),valporic acid,for-skolin,hydrocortisone,insulin,Triton,and sodium borohydride,were acquired from Sigma Chemical(St. Louis,MO).All monoclonal antibodies used in?ow cytometry analysis were purchased from BD Pharmin-gen(San Diego,CA).Rabbit anti-human neuron-speci?c enolase(NSE)antibodywas obtained from Polysciences(Warrington,PA).Goat anti-rabbit anti-bodyconjugated with Alexa Fluor488was from Molecular Probes(Eugene,OR).Bovine serum albumin (BSA)and normal goat serum were purchased from Jackson ImmunoResearch Laboratories(West Grove, PA).Paraformaldehyde solution was acquired from Electron MicroscopySciences(Fort Washington,PA). All tissue culture plates were BD Falcon t products. 2.2.Preparation of culture substrates

The expansion and neuron induction of human MSCs were performed in6?35mm multiwell tissue grade polystyrene plates.The surfaces of the wells were precoated with the substrate of interest at5m g/cm2 according to the manufacturer’s instruction.For Ma-trigel,higher coating density(50m g/cm2)was also tested. Unmodi?ed polystyrene surface served as a control.2.3.Expansion of MSCs

Frozen human MSCs(passage2)were thawed and plated on the substrates at5000cell/cm2.The cells were cultured in a-MEM supplemented with20%FBS,2m m L-glutamine,100U/ml penicillin,and100m g/ml strep-tomycin[5]at a volume-to-surface ratio of0.3.The medium was replenished after3–4days.Six days after plating,the cells were lifted byincubation with0.05% Trypsin and0.02%EDTA at room temperature for4–5min.Then the cells were washed,counted,and analyzed for phenotype.

2.4.Neuron induction

Two culture media were used to induce neuronal differentiation in MSCs[8].The preinduction medium contained DMEM,20%FBS,10ng/ml bFGF,100U/ ml penicillin,and100m g/ml streptomycin.The neuron induction medium consisted of DMEM,2%DMSO, 200m m BHA,25m m KCl,10m m forskolin,1m m hydro-cortisone,5m g/ml insulin,2m m valporic acid,100U/ml penicillin,and100m g/ml streptomycin.Frozen human MSCs(passage2)were thawed and plated on the substrates at5000cell/cm2and a volume-to-surface ratio of0.3.The cells were?rst cultured in DMEM+20% FBS for24h to allow recoveryfrom cry opreservation, and then in preinduction medium for24h.The preinduction medium was subsequentlyreplaced with equal volume of the induction medium.The cells were cultured for another24h before image analysis and immunocytochemical staining.For negative control, uninduced MSCs were maintained in DMEM+20% FBS on polystyrene for72h.

2.5.Flow cytometry

The phenotype of the expanded MSCs was analyzed using a FACSCalibur t system(BD Immunocytometry Systems,San Jose,CA).Brie?y,the harvested cells were incubated in DMEM+20%FBS at105–106cell/ml for 10–15min at room temperature to block nonspeci?c sites for antibodybinding.The cell suspension was added to a96-well U-bottom plate at100m l/well. Subsequently,10m l of antibodywas added to each well. Following30min of incubation at4 C in the dark,the cells were washed,?xed with1%paraformaldehyde at 200m l/well,and analyzed with?ow cytometry.

2.6.Immunocytochemistry

Immunocytochemical staining was performed to examine the NSE expression in MSCs at24h post neuron induction.The cultures were washed with D-PBS and?xed with4%paraformaldehyde at37 C for 10min.The cells were then permeabilized with1%

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Triton at room temperature for10min.Following2 rinses with0.5mg/ml sodium borohydride,the cells were incubated with5%normal goat serum and then6% BSA,each at room temperature for30min,to block non-speci?c antibodybinding.The cells were subse-quentlyincubated with a rabbit anti-human NSE antibodyat37 C for1h,and a goat anti-rabbit IgG antibodyconjugated with Alexa Fluor488at37 C for 40min.

2.7.Characterization of neuronal differentiation

Cell morphologywas monitored with an Oly mpus phase-contrast microscope.Images of the cultures were captured with a Nikon Coolpix990digital camera (Nikon-US,Melville,NY).The expression of NSE was examined using an Olympus BX-50?uorescence/DIC/ Phase Contrast microscope in conjunction with a Princeton Instruments PentaMAX cooled CCD camera

(Roper Scienti?c,Trenton,NJ)and the MetaMorph

Imaging System(Universal Imaging,Downingtown,

PA).The expression level in individual cells was

evaluated bymeasuring the mean?uorescent intensity

per pixel and the total intensityper cell after back-

ground subtraction.

Neuronal differentiation was quanti?ed based on5

non-overlapping images(10?)of each culture.The

percentage of cells displaying neuron-like morphology

in each image was calculated.The percentages of

neuron-like cells possessing secondaryand tertiary

branches,respectively,were also assessed.The mean

and the standard error of the mean(SEM)of the

measurements were then computed for each culture.

2.8.Data analysis

All experiments were repeated three times using

different lots of MSCs.All data are presented as mean 7SEM.Student’s t-test was used to evaluate the statistical signi?cance where indicated.Signi?cance level

was set at p o0:05:

3.Results

3.1.Expansion of MSCs

MSCs grown on all culture substrates displayed the

typical morphologies of undifferentiated MSCs.They

appeared to be small,round cells suspended in culture

medium when?rst plated.Within the initial24hours,a

majorityof the cells attached to the surface,elongated,

and spread to become spindle-shaped or large,?at cells.

However,cells cultured on poly-d-lysine were obviously

different in appearance:theywere less elongated and less spread out than on other substrates;cell densityalso appeared to be signi?cantlylower.

Following6days of expansion,?ow cytometry analysis indicated that over95%of the cells on each substrate expressed CD44but not CD34or CD45(data not shown),a phenotype typical of MSCs[1].The number of cells harvested was between0.6and2.3fold of that at initial plating and varied signi?cantlyamong experiments.To remove donor-to-donor variation,the expansion values were normalized based on the poly-styrene control within each experiment.Judging by the normalized expansion ef?ciency,there was no signi?cant difference in cell growth on surfaces coated with poly-l-lysine,collagen,laminin,?bronectin,and Matrigel compared with the polystyrene control.However, surfaces coated with poly-d-lysine supported markedly reduced cell proliferation,resulting in73%less expan-sion(Fig.1).

3.2.Neuronal differentiation of MSCs

Neuron induction resulted in an apparent morpholo-gical change in the culture on all substrates.The cells that were responsive to the induction displayed small, spherical,and refractive cell bodies.Theyalso developed long cellular processes,some of which formed second-ary,tertiary,and higher-order branches.In contrast,the unresponsive cells maintained the spread-out morphol-ogyobserved in cells without induction(Figs.2a–c).The differentiation status was veri?ed based on the expres-sion of human NSE,an enzyme found predominately in neurons and neuroendocrine tissues[14,15].Upon neuron induction,NSE expression was signi?cantly elevated in cells with contracted,spherical cell bodies and long processes compared with the spread-out cells, as indicated byhigher mean and total intensities of NSE

0.0

0.5

1.0

1.5

PS PDL PLL CO LN FN MG

Fig.1.The expansion ef?ciency of MSCs on unmodi?ed polystyrene (PS)or surfaces coated with5m g/cm2of poly-d-lysine(PDL),poly-l-lysine(PLL),collagen(COL),laminin(LN),?bronectin(FN),and Matrigel(MG).The expansion ef?ciencywas calculated as the ratio of the cell number at harvesting to that at initial plating,and was normalized based on the polystyrene control.The data represent the mean7SEM of three separate experiments.?indicates p o0:05 compared with the polystyrene control.

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staining (Figs.2d and e ).This enhancement was observed in the responsive cells on all substrates.

Neuron induction resulted in between 40%and 69%of neuron-like cells in the culture.The percentage of differentiated cells was similar on most substrates except for Matrigel at 5m g/cm 2,which led to 41%increase compared with the polystyrene control (Fig.3a ).The culture substrates also in?uenced the development of cellular processes,as judged bythe extent of process branching.The most extensive branching was again observed on Matrigel at 5m g/cm 2,on which the percentages of differentiated cells that possessed at least one secondarybranch or one tertiarybranch were 1.9and 6.8fold of the control,respectively.Although laminin also seemed to give rise to more extensive process branching,the difference was not found to be statisticallysigni?cant compared with the control.The poorest process development was seen on surfaces treated with poly-d -lysine,on which tertiary branching was not observed (Fig.3b ).3.3.Effect of coating density

Although Matrigel coating at 5m g/cm 2did not enhance the growth of MSCs,it improved the differ-entiation of the neuron-like cells resulted from neuron induction both quantitatively(as judged bypercentage)and qualitatively(as judged bythe improvement in high-order branching).To examine if further improvement could be attained at higher coating density,we tested culture surfaces with Matrigel coated at 50m g/cm 2.As shown in Fig.4a ,Matrigel at this densityincreased the expansion ef?ciencyby59%.The higher coating density

Fig.2.Neuron induction led to changes in cell morphologyand the expression of neuron-speci?c enolase (NSE)in MSCs.(a)Uninduced MSCs appeared as spindle-shaped or large,?at cells.10?magni?cation.(b)Cells that were responsive to neuron induction displayed spherical and refractive cell bodies and developed long,thin cellular processes (black arrow).In contrast,the unresponsive cells maintained the spread-out morphology(black arrow head).10?magni?cation.(c)Some of the responsive cells developed elaborate cellular processes with secondary(white arrow)and tertiary(white arrow head)branches.40?magni?cation.(d)Cells with undifferentiated morphologyexpressed onlylow level of NSE.(e)Upon neuron induction,NSE expression was signi?cantlyelevated in the responsive cells with contracted cell bodies and long processes.Scale bars=50m m (d–e).

0.0

20.0

40.0

60.0

80.0

% T o t a l C e l l s

(a)

0.0

20.0

40.0

60.0

80.0

PS PDL PLL CO LN FN MG 5

PDL PLL CO LN FN MG 5

% D i f f e r e n t i a t e d C e l l s

(b)

Fig.3.Neuronal differentiation of MSCs on unmodi?ed polystyrene (PS)or surfaces coated with 5m g/cm 2of poly-d -lysine (PDL),poly-l -lysine (PLL),collagen (CO),laminin (LN),?bronectin (FN),and Matrigel (MG5).The effect of the substrates was evaluated based on (a)the percentage of differentiated cells in culture after neuron induction;(b)the extent of cellular process formation characterized by the percentage of differentiated cells that displayed at least one secondarybranch or one tertiarybranch.The data represent mean 7SEM from one experiment and are representative of three separate experiments.?indicates p o 0:05compared with the poly-styrene control.

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also resulted in even more remarkable improvement in neuronal differentiation of MSCs.The percentage of neuron-like cells was 69%higher than the polystyrene control,as apposed to a 41%increase on Matrigel at 5m g/cm 2(Fig.4b ).Among the differentiated cells,the percentages of cells possessing secondaryand tertiary branches were 2.1and 9.8fold of the control,respectively(Fig.4c ).

4.Discussion

We observed that MSCs exhibited high growth potential on most of the tested substrates.This result corroborates previous reports on the high expansion capacityof MSCs [10,16].The onlyexception was observed with surfaces treated with poly-d -lysine,on which cell proliferation was severelylimited,possibly due to insuf?cient cell spreading [17].

Previous studies comparing the effect of polylysine,collagen,?bronectin,and laminin found that laminin

can most effectivelypromote neuronal differentiation and neurite outgrowth [18–21],possiblythrough speci?c interactions between its binding sites and cell surface receptors [22,23].In this study,we demonstrated that these substrates all supported the neuronal differentia-tion of MSCs,which do not normallydevelop into neural cell https://www.wendangku.net/doc/5113270041.html,pared with the polystyrene control,surfaces coated with laminin did not lead to obvious improvement in either the quantityor the morphologyof the differentiated cells.On the other hand,Matrigel gave rise to the most abundant MSC-derived neuron-like cells.These cells also exhibited the most extensive development of cellular processes,displaying a morphology that most resembled neurons.The major components of Matrigel are laminin,collagen type IV,entactin,and heparan sulfate proteoglycans [24],but it also contains various growth factors [25].It has been shown that Matrigel is more effective than its individual components,either alone or in combination,to stimulate axonal outgrowth [26].Previous studies also suggest that binding of growth factors to the

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0.5

1.0

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PS MG 5MG 50E x p a n s i o n E f f i c i e n c y

(a)

20.040.060.080.0

100.00.0

20.040.060.080.0100.0

PS MG 5MG 50

% T o t a l C e l l s

(b)

% D i f f e r e n t i a t e d C e l l s

Fig.4.The effect of surface coating densityon MSC expansion and neuronal differentiation.The effect was examined bycomparing Matrigel coated at 5m g/cm 2(MG5)and 50m g/cm 2(MG50)with unmodi?ed polystyrene (PS).(a)The expansion ef?ciency (normalized based on the polystyrene control).The data represent the mean 7SEM of three separate experiments.(b)The percentage of differentiated cells in culture after neuron induction.The values represent mean 7SEM from one experiment and are representative of three separate experiments.(c)The extent of cellular process formation characterized bythe percentage of differentiated cells that had at least one secondarybranch or one tertiarybranch.The data are mean 7SEM from one experiment and are representative of three separate experiments.?indicates p o 0:05compared with the polystyrene control.

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extracellular matrices not onlylocalizes these molecules to the immediate environment of the cells,but mayalso regulate their biological activities[27].Hence the enhancement we observed on Matrigel could be the concerted actions of its extracellular matrix components and inherent growth factors.

We also observed that increasing the coating density of Matrigel not onlyfurther enhanced the neuronal differentiation of MSCs but also resulted in improved cell expansion that was not seen on Matrigel at lower density.This could be due to increases in both the extracellular matrix proteins and the available growth factors.

Earlier studies showed that MSCs graduallylose their multiple-differentiation potential during cell expansion [16,28,29].Therefore,even though MSCs exhibit rapid growth,it is vital to propagate these cells maximally upon their isolation without further passages.Addition-ally,to achieve desired clinical ef?cacy,it is important to increase the yield of neuron-like cells from these expanded MSCs.Our?ndings suggest that the growth and neuronal differentiation of MSCs can both be signi?cantlyimproved through culture surface modi?ca-tion.Integration of our results implies a culture process that involves expanding and differentiating MSCs on Matrigel at50m g/cm2.Our?ndings mayalso encourage the development of novel biomaterials that mimic the chemical and physical microenvironment of neural tissues.Such materials can then be assembled with the MSC-derived neuron-like cells to form‘‘neo-tissues’’for transplantation.This approach has been shown to enhance the survival and function of neural transplants in other model systems[30].

5.Conclusion

In conclusion,our studies indicated that culture surface properties have signi?cant impact on the behaviors of MSCs.Matrigel coating at an appropriate densityprovides a favorable substrate that improves the ef?ciencyof cell expansion and neuronal differentiation. This?nding could contribute to the development of novel MSC-based cell transplantion and tissue engineer-ing applications for neural tissue repair and regenera-tion.Further investigation of the interactions between MSCs and Matrigel mayalso offer insights into the microenvironment that controls their neuronal differ-entiation.

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