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Mesenchymal Stem Cell and Chondrocyte Fates in a Multi-Shear Microdevice are

Regulated by Yes-Associated Protein

Weiliang Zhonga, Kang Tian

a, Linan Li

a, Weiguo Zhang

a*, Shouyu Wang

a*, Jianhua Qin

b

a Department of Orthopaedics, First Affiliated Hospital of Dalian Medical University, 222 Zhongshan Road,

Dalian, P.R. China

b Department of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian,

P.R. China

*Corresponding to Prof. Weiguo Zhang and Shouyu Wang

Tel/Fax: +86- 411-83632383

E-mail: weiguozhang63@gmail.com

Running Title: YAP and Mesenchymal Stem Cell and Chondrocyte Fates

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Abstract

Mechanical cues exert considerable influence on the fates of stem cells and terminally

differentiated chondrocytes. The elucidation of the interactions between cell fate and mechanical

cues in nuclear mechanotransduction will provide new clues to modulate tissue homeostasis and

regeneration. In this study, we used an integrated microfluidic perfusion device to simultaneously

generate multiple-parameter fluid shear stresses to investigate the role of fluid flow stimuli in the

regulation of YAP expression and the fates of MSCs and primary chondrocytes. YAP expression

was regulated by the level of fluid flow stimulus in both MSCs and chondrocytes. An increase in

the magnitude of stimulation enhanced the expression of YAP, ultimately resulting in an increase

in osteogenesis and a decrease in adipogenesis for MSCs, and initiating dedifferentiation for

chondrocytes. Cytochalasin D not only repressed nuclear YAP accumulation in the flow state but

also abrogated flow-induced effects on MSC differentiation and the chondrocyte phenotype,

resulting in MSC adipogenesis and the maintenance of the chondrocyte phenotype. Our findings

reveal the connection between YAP and MSC/chondrocyte fates in a fluid flow-induced

mechanical microenvironment and provide new insights into the mechanisms by which

mechanical cues regulates the cell fates of MSCs and chondrocytes.

Keywords: YAP; Microfluidics; Fluid shear stress; Mesenchymal stem cell; Chondrocyte

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1. Introduction

The regulation of stem cell differentiation and the chondrocyte phenotype remains a

challenge in tissue engineering and regenerative medicine. When the balance between the

adipogenesis and osteogenesis of mesenchymal stem cells (MSCs) and the maintenance of

chondrogenic phenotype are disrupted, bone and cartilage disorders such as osteoporosis and

osteoarthritis may arise [1]. During development, regeneration and homeostasis in the body,

mechanical cues play important role in regulating the fate and lineage commitment of stem cells

[2,3]. Therefore, it is important to elucidate the intricate reciprocal molecular interactions

between mechanical microenvironment and stem cells or chondrocytes in the musculoskeletal

system. Under physiological loading conditions, mechanical cues are recognized as an essential

component in the maintenance of articular cartilage matrix and healthy bone tissue homeostasis.

The interstitial flow driven by dynamic loading through the cartilage matrix or the canalicular

network of bone is an essential mechanical signal for the survival of chondrocyte or osteocyte

[4,5]. Increasing evidence has demonstrated that fluid flow plays an important role in regulating

cell behaviors and functions in the musculoskeletal system [6,7].

The cells perceive and transmit mechanical cues via mechanotransduction mechanisms. It

has been well established that the actin cytoskeleton plays a significant role in mechanosensing

and mechanotransduction [8,9]. The lineage commitment of MSCs can be predicted based on cell

geometry and cytoskeletal organization [10,11]. For example, flattened morphologies, stiffer

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substrates and tensile stress result in high cytoskeletal tension, which promotes osteogenesis. In

contrast, rounded morphologies, soft substrates and a lack of extrinsic mechanical force

contribute to adipogenesis [2,12]. Similarly, chondrocytes with a rounded shape or exposed to

soft substrates can maintain their phenotype; otherwise, these cells are likely to dedifferentiate

[13,14].

YAP (Yes-associated protein) plays crucial roles in cell proliferation, survival, differentiation,

tissue regeneration and organ size control, and these effects are mediated through the Hippo

pathway [15-17]. Recently, it has been confirmed that YAP is a regulator of the nuclear

transduction of mechanical cues [18]. The activity of YAP is regulated by intrinsically local cues,

such as cell shape [18,19], cell-cell contact [17,20], extracellular matrix stiffness [18] and

cytoskeleton tension [18,19,21], which have a distinct effect on the fate decision that occur

during stem cell differentiation [16,22-24]. However, the responses of YAP to extrinsic

mechanical force stimuli such as pressure stress, tensile stress and fluid shear stress have not been

well established. In particular, the relationships between YAP and the lineage commitment of

MSCs and chondrocyte phenotype in response to passive mechanical force remain incompletely

understood.

Currently, microfluidic-based microdevices are attracting much attention in the field of

cellular biomechanics, and their major advantage is the reduced time and resources they require

due to their increased experimental throughput and the minimized size of the experimental

platform compared with conventional way [25,26]. In this study, we employed a novel integrated

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microfluidic device that can simultaneously generate multiple shear stresses to investigate the

role of fluid flow stimuli in the regulation of YAP expression and the lineage commitment of

MSCs and chondrocytes.

2. Materials and methods

2.1. Microfluidic device design and fabrication

The design and operation of the microfluidic device were presented in Fig. 1A. The device

had one inlet, one outlet, and four cell culture chambers connected to resistance channels of

different dimensions. Each chamber was 100 µm in height, 1.2 mm in width, and 6 mm in length.

All devices were fabricated using conventional microfabrication techniques involving SU-8

(Microchem, Newton, MA, USA) photolithography and polydimethylsiloxane (PDMS) (Sylgard

184, Dow Corning, Midland, MI, USA) soft lithography [27]. Briefly, the mask was designed in

AutoCAD 2007 (Autodesk) and printed on transparencies with a 4000 dpi resolution. The

transparency mask was used in 1:1 contact photolithography with SU-8 photoresist to yield a

negative master that was constructed from a photoresist relief on a silicon wafer. The PDMS was

poured over the patterned wafer to completely cover the pattern, and the sample was placed in an

oven at 80°C for 60 min. The PDMS replica and a clean glass substrate were irreversibly sealed

using oxygen plasma (2 torr, 100 W) for 1 min.

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2.2. Numerical modeling

To evaluate the local fluid shear stress distribution in the chamber, we simulated the 3D flow

field in the chamber using a finite volume method (FVM) -based CFD code in FLUENT 6.3

(ANSYS, Inc., Lebanon, NH, USA). We used a model in which the flow was assumed to be

laminar, viscous, and incompressible, and we designed the microfluidic networks based on the

electric circuit analogy [28]. Using this analogy, individual channel sections were treated as

resistances within the flow circuit. A precise solution for the hydraulic resistance of the 3D

rectangular channel was derived via Fourier series expansions. To avoid the computational rigor

required to solve Fourier series expansions, we used an approximate version in algebraic form

[29]:

3

12 1

1 0.63( / )

LR

h w h w

(1)

where is the fluid viscosity, w is the channel width and h is the channel height, for h w .

For a square microchannel, the resistance can be calculated by

4

128.4R L

h (2)

A constant pressure drop p results in a constant flow rate Q . This result can be summarized

using the Hagen–Poiseuille equation, as follows [30]:

Hp QR (3)

Using simple algebraic manipulation, we determined the pressure values at the inlets and outlets

of the individual culture chambers. These pressure values were then used as the inlet and outlet

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pressure conditions to simulate the 3D flow field in each culture chamber using the CFD method.

The incompressible Navier-Stokes equations were used to model the steady-state flow field in the

culture chambers. It is worth noting that we did not consider the effect of the cells on the flows in

the present simulations. The computational domain was discretized using approximately 52,000

hexahedral meshes and solved using FVM along with the aforementioned inlet/outlet pressure

conditions and no-slip boundary conditions at the chamber walls. The density of the perfusion

medium was 993.2 kg/m3, and its viscosity was 7.85×10

-4 Pa s at 37°C.

2.3. The isolation and culture of MSCs and chondrocytes

All experimental procedures were approved by the Committee on Animal Use and Care of

Dalian Medical University. Articular cartilage chondrocytes were isolated from the

humeral heads, femoral heads and femoral condyles of male Sprague Dawley rats weighing

80-120 g, as previously described [31]. Briefly, chondrocytes were isolated by digestion with

0.15% type II collagenase for 16 h and resuspended in Dulbecco’s modified Eagle’s

medium/F-12 (Hyclone, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA), 50

mg/ml ascorbic acid-2-phosphate (Sigma, USA), and 100 units/ml penicillin-streptomycin. The

primary chondrocytes were used in the subsequent experiments. Primary rat mesenchymal stem

cells (MSCs) were isolated from the bilateral femurs and tibias of the same rats. The distal ends

of the bone were cut open, and the marrow cavities were lavaged with sterile phosphate-buffered

saline (PBS). The cells were resuspended in low-glucose Dulbecco’s modified Eagle medium

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(GIBCO Invitrogen, USA) containing 10% FBS (Hyclone, USA) and 100 units/ml

penicillin-streptomycin. After 48 h of incubation at 37°C in 5% CO2, the medium was changed to

remove the non-adherent cells. After two passages, the attached MSCs were devoid of any

non-adhering cells and used in the following experiments.

The microdevice was sterilized in an autoclave and then air-dried on a clean bench. The cell

culture chambers were coated with 100 μg/ml fibronectin (Sigma, USA) for 1 h at room

temperature. Then, the chambers were washed with PBS. MSC suspension of 0.5×105 cells/ml

and a chondrocyte suspension of 1×105 cells/ml were individually injected into chambers through

the outlet, and the device was incubated at 37°C for 12 h to allow cell attachment. Then, the inlet

of the device was connected to a peristaltic pump (Longer Pump BT100-2J, China), and the outlet

was connected to a reservoir. MSCs were exposed to adipogenic-osteogenic co-induction medium

during perfusion culture. The mixed induction medium contained 1:1 adipogenic

induction:osteogenic differentiation media (Cyagen Biosciences Inc, Sunnyvale, CA, USA), as

previously described [12]. The cells were exposed to 1 µM cytochalasin D (CytoD) (Sigma, USA)

for 1 h to disrupt stress fibers before the application of the flow stimulus.

2.4. Immunofluorescence staining

Samples in the device were washed with PBS, fixed with 4% paraformaldehyde at room

temperature for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After washing

with PBS 3 times, the samples were blocked with normal goat serum at room temperature for 30

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min, incubated with primary antibodies against YAP (Santa Cruz, USA), PPAR (Santa Cruz,

USA), Runx2 (Santa Cruz, USA), Sox9 (Santa Cruz, USA), collagen II (Sigma, USA), collagen I

(Sigma, USA) at 4°C overnight; and then incubated with FITC-conjugated goat anti-rabbit IgG or

TRITC-conjugated goat anti-rabbit secondary antibodies (Zhongshan, China) at room

temperature for 1 h. The nucleus was stained with 4,6-diamino-2-phenyl indole (DAPI)

(Invitrogen, USA) for 10 min. After staining, the devices were washed with PBS 2-3 times and

imaged using fluorescence microscopy (Olympus IX71). The fluorescence intensities of collagen

I and collagen II were determined from the fluorescence photographs (N=10) using Image-Pro

Plus 6.0 software to obtain relative fluorescence intensity (RFI) values.

2.5. Cell staining

After 5 days of perfusion, the cells were stained with oil red O and alkaline phosphatase

(ALP) to determine adipogenic differentiation and osteogenic differentiation, respectively. Briefly,

the cells were fixed with 4% paraformaldehyde and then stained with Fast Blue RR/naphthol

(Sigma, USA) to visualize ALP. After washing with PBS, the cells were stained with 30 mg/ml

oil red O (Sigma, USA) in 60% isopropanol to visualize the lipid droplets and then rinsed in PBS.

2.6. Statistics and analysis

All experiments were performed at least in triplicate with different batches of devices.

Differences among three groups were analyzed using one-way ANOVA. Comparisons of two

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groups were performed by using the two-sample t-test. P<0.05 was considered statistically

significant.

3. Results

3.1. Computational simulation of the fluid dynamics in the microchambers

In this study, a microdevice was designed based on the principles of the Electric Circuit

Analogy. This device was composed of one inlet, one outlet, resistance channels and four cell

culture chambers. The flow of fluid through the microdevice was typically driven by a peristaltic

pump. Thus, the pressure-driven fluid flow through the chamber could be defined as steady-state

flow and modeled as Poiseuille flow. The fluid shear stress on the cells could be assumed to be

equal to the wall shear stress at the bottom of chamber. When the perfusion flow rate of 30

µl/min was applied to the microdevice, different levels of fluid flow stimulus could be generated.

The central area of these chambers exhibited a uniform distribution for the wall shear stress (Fig.

1B). The results demonstrated that the majority of the bottom of the chamber experienced

uniform shear stress except the areas near the corners, the inlet, the outlet, and the side walls. To

quantify the local distribution of shear stress in detail, we computed the wall shear stress along

the vertical and horizontal center axes of each microchamber. This observation was further

confirmed by the wall shear stress curves along the vertical center axis (Fig. 1C) and horizontal

center axis (Fig. 1D). The average bottom wall shear stresses in Chamber 1 to Chamber 4 in these

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uniform regions were 1.089 dyne/cm2, 0.231 dyne/cm

2, 0.055 dyne/cm

2, and 0.009 dyne/cm

2,

respectively.

3.2. The proliferation rates of MSCs and chondrocytes exposed to different shear stresses

To examine the effects of different shear stresses on cell proliferation, the proliferation rates

of MSCs and chondrocytes were analyzed by counting the number of cells present in the

chambers under each condition. As shown in Fig. 2A, the proliferation rate of MSCs increased

significantly in response to shear stress at 0.231 and 1.089 dyne/cm2 after 24 h and 48 h of

perfusion compared to the minimum stimulus (0.009 dyne/cm2). For chondrocytes, the

proliferation rate increased significantly in response to shear stress at 1.089 dyne/cm2 after 24 h

and shear stress at 0.231 and 1.089 dyne/cm2 after 48 h. These findings suggested that the

proliferation rates of both MSCs and chondrocytes increased with an increasing magnitude of the

fluid shear stress.

3.3. The changes in YAP expression in response to different shear stresses

To investigate the effects of different fluid shear stresses on YAP expression in MSCs and

chondrocytes, the cells in the microdevices were stained for YAP after 5 days of perfusion for

MSCs and 2 days of perfusion for chondrocytes. As shown in Fig. 2B, the distribution of YAP in

MSCs was mainly in the nucleus with low level in the cytoplasm. Although the increase in fluid

shear stress did not alter YAP localization, the level of YAP in the nucleus tended to increase (Fig.

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2B). In addition, we found that in primary chondrocytes YAP was predominantly cytoplasmic at

an extremely low level of shear stress. However, increased nuclear YAP accumulation was

observed with an increasing magnitude of stimulation. At higher stimulus levels (0.231 and 1.089

dyne/cm2), the nuclear localization of YAP was markedly increased (Fig. 2B). Conversely, MSCs

and chondrocytes treated with CytoD developed a more rounded morphology and exhibited YAP

diffusion into the cytoplasm after exposure to the fluid flow. The quantification of nuclear YAP in

MSCs and chondrocytes confirmed these findings (Fig. 2C). The percentage of nuclear YAP in

these two types of cells was significantly higher under shear stresses of 0.231 and 1.089 dyne/cm2

than under the lowest level of shear stress (0.009 dyne/cm2). CytoD treatment was associated

with a significant decrease in nuclear YAP relative to the lowest stimulus level.

3.4. The fate of MSCs changes under different shear stress conditions

To evaluate the effects of different fluid shear stresses on MSC differentiation, cells in the

microdevices were stained for ALP and intracellular lipid droplets after 5 days of perfusion. As

shown in Fig. 3A, cells exposed to an extremely low level of shear stress tended to undergo

adipogenic differentiation, but with an increasing magnitude of shear stress, increased cells

underwent osteogenic differentiation. Especially at the higher shear stress level of 1.089

dyne/cm2, cells tended to become committed to the osteogenic lineage. The ratio of osteogenic

commitment to adipogenic commitment reflected a significant mechanical effect of different fluid

shear stresses (Fig. 3B). However, CytoD-treated cells exposed to the fluid flow stimulus

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appeared to lose the ability to undergo osteogenic differentiation regardless of the level of flow.

Interestingly, the pattern of adipogenic and osteogenic differentiation were consistent with the

pattern of YAP distribution in the cytoplasm and nucleus.

Next, we examined the expression of more lineage-specific regulators such as PPAR,

Runx2, and Sox9 in MSCs. As shown in Fig. 3C, PPAR that promotes adipocyte differentiation

in MSCs was predominantly localized in the nucleus, but a gradual decline in PPAR expression

was observed with an increasing magnitude of flow stimulus. In contrast, Runx2, a key regulator

of osteogenic differentiation, displayed a gradually increasing expression in the cells, especially

in the nucleus with an increasing magnitude of flow stimulus. However, CytoD-treated cells

displayed high expression level of PPAR but low expression level of Runx2 regardless of flow

stimulus. For Sox9, an essential regulator of chondrogenesis, no obvious expression was detected

under different fluid shear stress even when treated with CytoD. Taken together, we found that

the changes in the expression of PPAR and Runx2 were in accordance with the variation in lipid

droplets and ALP.

3.5. The phenotypic variations of chondrocytes in response to different shear stresses

After 2 days of fluid flow stimulation, the phenotypic changes in primary chondrocytes were

investigated by immunofluorescence staining for collagen I and collagen II. As shown in Fig. 4A,

primary chondrocytes were nearly negative for collagen I and positive for collagen II when

exposed to extremely low level of fluid flow. However, with an increasing magnitude of shear

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stress, the cells began to synthesize collagen I, whereas the expression of collagen II diminished.

When the cells were treated with CytoD before the exposure to the fluid flow stimulus, the

hyaline cartilage phenotype of the cells was rescued, with low expression of collagen I. To further

quantify the variation in the expression levels of collagen II and collagen I, the relative

fluorescence intensities were plotted (Fig. 4B and C). We found that from 0.055 to 1.089

dyne/cm2, RFI values were significantly higher for collagen I and lower for collagen II than the

values at the minimum level of stimulus. As shown in Fig. 2 and Fig. 4, we found that

chondrocyte dedifferentiation was concomitant with YAP accumulation in the nucleus, indicating

that mechanical cue of fluid flow leads to increased YAP expression in dedifferentiated

chondrocytes.

4. Discussion

YAP transcriptional coactivator has emerged as a key mediator of the regulation of cell

behaviors such as proliferation, survival, apoptosis and differentiation [16,17,21]. In this study,

we evaluated the change in YAP expression and the consequent biological responses to

flow-induced mechanical signals of different magnitudes. Our results suggest that YAP may

mediate the effects of fluid flow-induced shear stress on the fates of MSCs and chondrocytes.

We characterized the changes in YAP expression along with the fates of MSCs and

chondrocytes in response to different levels of flow stimulus. Our data showed that YAP

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expression in MSCs was increased with an increasing magnitude of shear stress. Furthermore, we

demonstrated that in chondrocytes YAP was translocated into the nucleus in response to fluid

flow stimulus. The transduction of local mechanostimuli into biochemical signals occurs through

several signaling pathways. Recently, the transcriptional regulators YAP/TAZ have been shown

to act as not only sensors but also mediators of physical signals, shuttling between the cytoplasm

and the nucleus [22]. As the downstream effectors of mechanotransduction, YAP function

depends on the tension of the actomyosin cytoskeleton and Rho GTPase activity because proper

cytoskeleton tension is necessary to maintain YAP transcriptional activity. Moreover, increasing

evidence has shown that shear stress can cause cytoskeleton reorganization in mechanically

sensitive cells. Thus, the flow-dependent changes in YAP expression are likely due to an increase

in cytoskeleton tension. When the flow shear force is transmitted to the intracellular space, the

cells re-organize the cytoskeleton to gradually increase the strength of attachment with an

increasing magnitude of stimulation. Meanwhile, cytoskeletal cues can mediate YAP nuclear

localization to ultimately affect gene expression. It is well known that the actin cytoskeleton is

disrupted when cells are treated with CytoD, thereby abrogating the ability of cells to sense

external mechanical cues. In this study, the treatment of cells with CytoD resulted in YAP

downregulation, and the flow-dependent response was abolished, further suggesting that shear

flow plays a role in the regulation of YAP and the integrity of the actomyosin cytoskeleton is

vital for shear stress-based regulation of YAP expression. Additionally, the effects of fluid shear

stress on cells involve other pathways that may communicate with YAP or Hippo pathway. The

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cell-cell contact or a high cell density inhibits YAP activity by activating the Hippo pathway [20].

To avoid such an influence, a low seeding density was employed in our microdevice experiments.

We assumed that this may limit the activation of Hippo pathway and better investigate the effects

of fluid shear stress on YAP. Consequently, we found that an increasing magnitude of fluid shear

stress could promote cell proliferation and concomitantly enhance YAP expression in the nuclei.

Shear stress-induced proliferation of osteocytes, MSCs, as well as chondrocytes has been

reported [32-34], involving several mechanisms such as ERK pathway, MAP kinase pathway,

NO/cGMP/PKG and calcium signaling [35]. In addition，mechanical signaling through the

cytoskeleton linkage between focal adhesion and regulators of cellular contractility contribute to

the regulation of cell proliferation [36]. YAP has been shown to act as a transcriptional

co-activator of TEAD transcription factors to promote cell proliferation and survival in many

tissues [37]. Here, we propose that fluid flow upregulates YAP, which then promotes cell

proliferation by binding to the TEAD family of transcription factors. Therefore, we conclude that

nuclear YAP expression is a key regulator that has a correlation with mechanical signal-induced

proliferation. Further investigations are required to determine whether these known pathways

cooperate with the Hippo/YAP pathway to promote cell proliferation.

Control of the balance between adipogenesis and osteogenesis during MSC differentiation is

necessary to maintain bone homeostasis. Stem cells are highly sensitive to mechanical cues and

can convert mechanical stimuli into biochemical signals via mechanotransduction systems. In this

study, we characterized the effects of distinct levels of flow stimulus on the regulation of MSC

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fate. We found that the expression level of YAP is correlated with the intensity of the stimulus

experienced by the cells and the fates of the MSCs. Increased YAP expression is in accordance

with the increase in the expression of Runx2 and ALP, indicating that increased YAP expression

may contribute to osteogenesis in MSCs. Conversely, decreased YAP expression is concomitant

with the increase in lipid droplets and the expression of PPAR, which indicate adipogenesis in

MSCs. It has been reported that YAP interacts with Runx2 and PPARγ to regulate

adipocyte/osteocyte gene expression [38]. Moreover, YAP modulated Runx2 and PPARγ activity

by altering the activation of Wnt/β-catenin signaling [39]. It is therefore possible that fluid shear

stress regulates the choice between adipogenesis and osteogenesis in MSCs by controlling the

expression of YAP. Growing evidence has shown that the enhancement of mechanical or

cytoskeletal cues would increase osteogenic differentiation and decrease adipogenic

differentiation. In addition, cell density is an important factor that regulates the adipogenic and

osteogenic differentiation of MSCs [12,40].

In a multilineage differentiation experiment, MSCs differentiated into adipocytes in

response to induction medium containing 10-6

M dexamethasone but differentiated into osteocytes

in response to induction medium containing 10-7

M dexamethasone [41]. In this study, the mixed

differentiation medium contained a relatively high concentration of dexamethasone because the

two types of induction medium were mixed in equal proportions, thus causing an increase in the

proportion of cells undergoing adipogenic differentiation. However, with increases in the shear

stress magnitude, the proportions of cells undergoing adipogenic and osteogenic differentiation

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were reversed due to the upregulation of YAP expression, leading to an increase in osteogenesis

and a decline in adipogenesis. Thus, by analyzing YAP expression level, we can preliminarily

estimate the lineage to which the MSCs have committed or will commit and determine which

flow stimulus condition is appropriate for target differentiation.

Chondrocytes differentiate from mesenchymal cells during development and are apt to

dedifferentiate in in vitro cultures [42]. Excess fluid flow stimulus has been shown to be

detrimental to the maintenance of chondrocyte function [43-45]. We first investigated the

expression of YAP in response to distinct levels of fluid flow in primary chondrocytes and then

monitored the changes in the chondrocyte phenotype that are associated with the changes in YAP

localization. We found that increasing flow stimulus resulted in nuclear YAP accumulation and

the loss of chondrocyte properties. When primary chondrocytes were exposed to extremely low

stimulus, no phenotypic variation was observed compared to the higher stimulus, suggesting that

such a condition is not enough to induce chondrocyte dedifferentiation. Instead, this condition

apparently mimicked the flow of interstitial fluid in the cartilage and played an important role in

nutrient transport. A previous study confirmed that YAP expression was suppressed by treatment

with CytoD [19]. In our study, after the disruption of the cytoskeleton with CytoD, the cells

undergoing YAP cytoplasmic translocation failed to respond to the flow stimulation but better

maintained their phenotype, suggesting that the process of regulating the chondrocyte phenotype

is related to YAP expression. Taken together, our findings indicate that the maintenance of the

phenotype of primary chondrocytes is associated with the exclusion of YAP from the nucleus,

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which may involve mechanical and cytoskeletal cues or the Hippo pathway. For instance, both

pellet or monolayer culture with high cell density and soft substrate with low cytoskeleton

tension can better maintain chondrocyte phenotype in vitro. Such a high cell density induces the

activation of Hippo signaling to suppress the expression of YAP [20], and low cytoskeleton

tension also causes YAP inactivation [18]. The detailed biochemical mechanisms underlying

these phenomena await further characterization. Notably, based on the localization of YAP in

chondrocytes, we can estimate the stimulus intensity experienced by cells and evaluate whether

chondrocytes would dedifferentiate when exposed to a particular magnitudes of shear flow.

Microtechnology can be used to regulate biological stimuli at the cellular and subcellular

scales, enabling us to decipher the roles of specific cues and mechanisms in the cells [46]. In our

study, we combined the electric circuit theory with CFD analysis to design a lab-on-a-chip and

thus solve the problems related to complex microfluidic network-based devices. Using a physical

analogy, the systematic networking of the microchannels from the inlet to outlet ports could

readily be used to establish relatively simple model. Thus, multiple shear stresses could be

obtained through the different inlet channel resistances of the different chambers. Furthermore,

this microdevice allowed the control of multiple shear stress values in a particular range by

modifying the inlet resistance of each microchamber and the inlet flow rate. In addition, this

microdevice provided a simple platform to rapidly screen cellular responses to different levels of

flow stimulus and minimize the sample volume to the micron scale.

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5. Conclusions

Using microfluidic techniques, we developed a novel integrated microdevice that

simultaneously produces multiple fluid shear stresses comparable to or stronger than that of

interstitial flow. Using this device, the roles of the fluid flow stimulus in the regulation of YAP

expression and the fates of MSC and chondrocytes were investigated. Our results suggest that

YAP expression in MSCs and chondrocytes is regulated by fluid shear stress and YAP mediates

the fate determination of MSCs and chondrocytes in response to fluid flow stimulus. Therefore,

the modulation of YAP expression could be exploited to manipulate stem cell fate and increase

the regenerative potential of terminally differentiated organs with poor intrinsic regenerative

capacity.

Acknowledgements

This work was supported by the National Nature Science Foundation of China (No.

81171464 and No. 81270052). These authors have no conflict of interest.

Author Disclosure Statement

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No competing financial interests exist in this study.

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Figure Legends

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Page 28 of 35

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Fig. 1. A: Schematic diagram of the multi-shear microfluidic device and a photograph of a

prototype microdevice. In the schematic diagram, Q is the flow rate in the microchamber, w is the

width of the microchamber and h is the height of the microchamber. B-D: The numerical

simulation results for the cell culture chambers based on CFD analysis. B: Contours of the wall

shear stress distribution in the microchambers of the device. C: Variation in the wall shear stress

along the vertical center axis of each microchamber. D: Variation in the wall shear stress along

the horizontal center axis of each microchamber.

Page 29 of 35

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Page 30 of 35

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Fig. 2. A: The proliferation of MSCs and chondrocytes in the presence of different shear stresses.

(i): Histogram of MSC proliferation rates after 24 and 48 h. (ii): Histogram of chondrocyte

proliferation rates after 24 and 48 h. The values are the mean ± SD. * and # represent 24 and 48

hours, respectively. #

and *, P<0.05; ##

and **, P<0.01 versus 0.009 dyne/cm2. B-C: The changes

in YAP expression in cells subjected to different shear stresses. MSCs and chondrocytes were

immunostained for YAP (green) after 5 and 2 days of perfusion, respectively. B: The YAP

distribution in MSCs and chondrocytes subjected to different shear stresses. Scale bar: 20 μm. C:

Graphs indicating the percentage of cells with nuclear YAP under different conditions. The values

are the mean ± SD. * and # represent MSCs and chondrocytes, respectively.

# and *, P<0.05;

###

and ***, P<0.001 versus 0.009 dyne/cm2.

Page 31 of 35

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Page 32 of 35

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Fig. 3. MSC commitment varies with changes in the flow stimulus. A: Brightfield images of the

adipogenic and osteogenic differentiation of MSCs after 5 days of exposure to different induction

conditions. The cells were stained with Fast Blue RR/naphthol and oil Red O to visualize ALP

(blue) and lipid droplets (red) as indicators of osteogenesis and adipogenesis, respectively. Scale

bar: 100 μm. B: Percentage of osteogenic and adipogenic differentiation of MSCs after exposure

to the mixed induction medium for 5 days. The error bars are mean ± SD. C: Comparison of the

expressions of lineage-specific regulators (PPAR, Runx2 and Sox9) in MSCs under different

induction conditions. Fluorescence images of PPAR, Runx2 and Sox9 staining in cells exposed

to different conditions for 5 days. Scale bar: 20 μm.

Page 33 of 35

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Fig. 4. The phenotypic variations of chondrocytes in response to different shear stresses.

Chondrocytes were immunostained for collagen I (green), collagen II (red), and DAPI (blue) after

2 days of perfusion. A: Fluorescence images of collagen I and collagen II staining in cells

exposed to different conditions. Scale bar: 50 μm. B: Quantitative analysis of the relative

fluorescence intensities of collagen II and collagen I under different conditions. The error bars are

the mean ± SD. *P < 0.05 versus 0.009 dyne/cm2.

Page 34 of 35

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Page 35 of 35

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