Organizer: International Research
Center for Wound Repair and
Regeneration, NCKU
The schedule of 2013 International Symposium on Wound Regeneration and Repair
2013 年 10 月 8 日(星期二) Tuesday, Oct 8 2013
成功大學圖書館 B1 國際會議廳 (台南市大學路一號)
National Cheng Kung University (No.1, University Road, Tainan City 701, Taiwan R.O.C.)
Time 時間
08:30-08:40
08:40-08:50
08:50-09:20
09:20-09:50
09:50-10:05
10:05-10:20
10:20-10:40
10:40-11:00
11:00-11:30
11:30-11:45
11:45-12:00
12:00-12:15
12:15-13:30
13:30-14:00
14:00-14:15
14:15-14:30
14:30-14:45
14:45-15:00
15:00-15:30
Topic 主題
President Huang and Dean Chang
Opening Remarks (開幕致詞)
Speaker 講者
Prof. Cheng-Ming,
Chuong
Regulation of limb morphogenesis by Prof. David Gardiner
information in the ECM
Regeneration of limbs
Prof. Jonathan Slack
Clinical strategies and aspects in
Shyh-Jou Shieh
wound repair and regeneration
Roles of ABCG2 transporter in stem Chia-Ning Shen
cells, liver metabolism and
transdifferentiation
Developing co-culture microfluidic
Chia-Ching(Josh) Wu
system for stem cell and tissue
regeneration
Coffee Break
Mechanism of skin pattern
Prof. Shigeru Kondo
regeneration in zebrafish
Mechanotransduction for Keloid
Ming-JerTang
fibroblast
Collagen and hyaluronan for
Lynn LH Huang
regenerative medicine
Epigenetic modulation of tissue
Michael Hughes
regeneration
Lunch / Poster Discussion
The dedifferentiation and
Prof. Ken Muneoka
transdifferentiation of perivascular
fibroblasts during digit tip
regeneration in mice
Developmentally-regulated skin
Tai-lanTuan
wound repair
Thrombomodulin regulates
Hua-Lin Wu
keratinocyte differentiation and
(Tsung-Lin Cheng)
promotes wound healing
Topology-dependent hair plucking
Chih-Chiang Chen
leads to non-linear amplification of
regenerative response via a
distress-gauging cellular circuit
Coffee Break
Complex tissue regeneration in a new Prof. Malcolm Maden
mammalian model system, the
African spiny mouse
Moderator 主持人
President
Ming-JerTang
Vice Dean
Ming-Der Lai
Prof. Hua-Lin Wu
Director
Chi-Chang Shieh
15:30-15:45
15:45-16:00
16:00-16:15
16:15-16:45
16:45-17:15
17:30
Mechanics-driven Self-organization
in Tissue-scale Tubulogenesis
Towards a cell based strategy of
nipple regeneration for mastectomy
victims
The regeneration of feathers after
different levels of wounding
Chinlin Guo
John Foley
Ting-Xin Jiang
Selected abstract for oral presentation Wei-Ling Lin
Dr. Shyh-Jou
Yuan-Yu Hsueh
Shieh/Michael
Hans Harn
Hughes/Josh Wu
Discussion on identifying solvable
Dr. Chuong, Shieh, Hughes, and panel
important issues
members
Close
The location of symposium room
Abstract
大會演講摘要
Morphogenesis in Development and Regeneration
Cheng-Ming, Chuong
University of Southern California
To accomplish the goal of regenerative medicine, we need to obtain stem cells, guide the stem cells to form
tissues / organs we desire, and to deliver the products to patients properly. There has been good progress in
obtaining stem cells in the form of ES and iPS cells. There also has been good progress in guiding stem cells
to differentiate into certain cell types . However, scientists still have a lot to learn about how to make
morphology, the science of morphogenesis. Some investigators use scaffolds to engineer tissue shapes, but
soon find that the cells may not maintain the engineered structure for long. Since stem cells undergo
morphogenesis to make tissues and build organs during development, it would be ideal to elicit the
self-patterning, self-organizing potential of cell populations with minimal engineered effort applied only at
key steps. To do this, we need to understand the fundamental principles of morphogenesis used in
development and regeneration.
To this end, my team has been using skin appendages as the Rosetta stone to decipher the language of
morphogenesis. This is because feathers and hairs undergo regeneration in physiological conditions, where
stem cells produce new phenotypes in different body regions ideal for their function at different stages of the
organism’s life (Chuong eta l., 2012, 2013).
By studying feather regeneration, we have found modulating
the balance of morphogenesis related molecules can make different feather patterns (Lin et al., 2013a, b). By
studying regenerative behavior of the hair population, we found that the extra-follicular macro-environment
can modulate hair stem cell activity, mediating changes in body hormone status (Plikus et al., 2008, 2011;
Chen and Chuong, 2012). Using a genetic approach, we found epigenetic enzymes are essential for
morphogenetic activities and are working on their genomic changes (Li et al., 2013; Hughes et al., 2013).
The progress will be reported by my team members and collaborators.
Together with investigators from Taiwan, we are glad this meeting offers the opportunity to exchange our
findings with renowned international scholars in the study of morphogenesis. Some study the development
of different organs, and some study animals with unusually robust regenerative abilities. We hope to have
an exciting interactive symposium to catalyze new understanding and inspire new approaches to the study of
morphogenesis in development and regeneration.
Regulation of limb morphogenesis by information in the ECM
David M. Gardiner
University of California Irvine
Successful regeneration requires replacement of both structure and information. The need for the former is
obvious since lost structure needs to be replaced. The structure is rebuilt by cells, and thus the focus of
attention of almost all regeneration research has been on how undifferentiated cells (e.g. stem cells) can be
induced to differentiate into the desired cell type. The need for information is not so obvious, but is critical
nevertheless. The goal of regeneration is the restoration of function, which is an emergent property of the
structures that are reformed. For example, in the limb, the regenerated muscles must be coordinately
patterned relative to the joints and long bones to allow for flexion and extension, and the regenerated nerves
must be patterned so as to coordinately innervate the muscles. The fact that information exists and is a
property of specialized cells is most evident from studies of animals that can regenerate, and may be the
most important insight to be gained from such studies. These specialized pattern formation cells are
localized within the connective tissue where they form an information “grid” that wraps around all the
organs and underlines the skin of the body. It likely will be a long time before we learn how to repair this
information grid endogenously like what happens during salamander limb regeneration. Nevertheless, it is
important that we recognize that it exists, and make efforts to better understand how it is established during
development and reestablished during regeneration. We have come to realize that the information of the grid
is at least in part encoded by specifically-regulated modifications of the extracellular matrix (ECM). We
thus can use the knowledge and insights about the nature and function of the grid gained from understanding
salamander limb regeneration as a guide to modify the ECM and regulate growth and pattern formation
during regeneration.
Reprogramming of liver cells toward a beta cell phenotype using transcription factors
J.M.W.Slack
University of Minnesota
In embryonic development, the pancreas and liver share their developmental history up to the stage of bud
formation. Because of this we postulated that direct reprogramming of liver to pancreatic cell types may be
possible when suitable transcription factors are overexpressed.
Using a polycistronic adenovector we have investigated the overexpression of Pdx1, Ngn3 and MafA in
various cell types. Upregulation of insulin and other beta cell genes is most pronounced in pancreatic
exocrine cells, then in liver-derived cells, and least in fibroblasts, supporting the idea of susceptibility
depending on developmental relatedness. Moreover, embryonic hepatoblasts are more easily transformed
than postnatal hepatocytes. In these transformations, although a wide range of characteristic beta cell genes
become active, the target cells are not reprogrammed to a full beta cell phenotype.
The most interesting results are obtained when the three genes are administered in vivo to mice rendered
diabetic by treatment with streptozotocin. The diabetes is relieved long term and many ectopic duct-like
structures appear in the liver which express beta cell markers and secrete insulin in response to external
glucose. Lineage labeling shows that the target cells are SOX9-positive and may be epithelial cells of small
bile ducts or hepatoblast-like progenitor cells found in the periportal region. This reprogramming method
works either with immunodeficient (NOD-SCID) mice, or with normal (CD1) mice if they are also treated
with a PPAR agonist, which increases cell division in the SOX9- positive cell population. The
insulin-secreting ducts persist long after the viral gene expression has ceased, indicating that this is a true
irreversible cell reprogramming event.
Roles of ABCG2 transporter in stem cells, liver metabolism and transdifferentiation
Chia-Ning Shen
Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
The ATP binding cassette transporter ABCG2 which has been used widely as a stem cell marker is
found not only to express in a wide variety of stem cells but also in several somatic tissues including liver.
My lab has been trying to determine molecular basis of the transdifferentiation that occurs naturally. The
appearance of hepatic foci in pancreas is well-documented both in animal experiments and in cases of
pancreatic cancer. We initially demonstrated ABCG2-positive intermediates derived from acinar-to-hepatic
transdifferentiation were multipotent. Treatment of ABCG2 inhibitors can suppress acinar-to-hepatic
transdifferentiation. To explore the role of ABCG2 in pancreas further, we analyzed the pancreas repair after
cearulien-induced acute pancreatitis. Initially, we found CK19-positive ductal cells were increased in
pancreatitis in both wild-type and ABCG2 knock mice. However, reduction in ductal cells was only seen in
wild type mice accompanied by acinar tissue regeneration. In ABCG2 KO mice, we not only found
appearance of CK19-positive ductal cells in central acinar area, but we also found delay in acinar
morphogenesis. The delay is wound repairing was also found in the skin of ABCG2 knockout mice. We
further revealed that ABCG2 deficiency impaired re-epithelization as judge by pre-matured expression
CK10 in CK14-positive basal cells was observed in the suprabasal layer of the epidermis. Transplantation
studies further showed that knockout of ABCG2 in epidermal stem cells will affect the differentiation
potential of epidermal stem cells to either keratinocytes or to hair follicle cell lineages. To further determine
the physiological role of ABCG2 transporters in stem cells and in somatic tissue, ABCG2 knockdown and
ABCG2 knockout was carried out in embryonic stem cells and adult mouse liver. We demonstrated that (1)
ABCG2 was found to directly involve in regulating protoporphyrin IX homeostasis which prevents
undifferentiated ES cells to excess oxidative stresses. We also found p53 played as a downstream
checkpoint of ABCG2-dependent defense machinery in order for maintaining the genetic integrity of ES
cells; (2) ABCG2-deficient hepatocytes had increased amounts of fragmental mitochondria accompanied by
disruption of mitochondrial dynamics and functions. And this disruption was due to ABCG2 knockout
elevating intracellular protoporphyrin IX led to upregulation of DRP-1-mediated mitochondrial fission. And
this upregulation of DRP-1 was induced by activation of p53 pathway as knockout of p53 can restore the
mitochondrial fusion phenotype in ABCG2-deficient hepatocytes. Taken together, the finding that ABCG2
deficiency can affect repairing of skin wound and pancreatic injury and may generate dysfunctional
mitochondria in hepatocytes should raises concerns regarding the systematic use of ABCG2 inhibitor in
patients.
Developing co-culture microfluidic system for stem cell and tissue regeneration
Chia-Ching Wu
National Cheng Kung University
The aim of this study is the functionalization of kidney epithelial cells with capsule-like constitution in a
coculture microfluidic device. Current hemodialysis has functional limitations and is insufficient for renal
transplantation. The bioartificial tubule device has been developed to contribute to metabolic functions by
implanting renal epithelial cells into hollow tubes and showed higher survival rate in acute kidney injury
patients. In healthy kidney, epithelial cells are surrounded by various types of cells that interact with
extracellular matrices which are primarily composed of laminin and collagen. The current study developed a
microfluidic coculture platform for bioartificial renal chips with multiple microfluidics channels that are
microfabricated by polydimethylsiloxane. The feature of Bowman's capsule in the nephron was
manufactured for the proposed coculture microfluidic device. The coculture device used adipose-derived
stem cells (ASCs) and epithelial cell to demonstrate the epithelization under cell-cell interaction. This
bioartificial renal chip proved the possibility to provide fluid exchange in current microfluidic devices
without damaging the coculture structure or cells. Collagen gel (CG) encapsulated with adipose-derived
stem cells (CG-ASCs) was injected into a central microfluidic channel for three-dimensional (3D) culture.
The resuspended Madin–Darby canine kidney (MDCK) cells were injected into nascent channels and
formed an epithelial monolayer. This coculture microfluidics device allowed real-time monitoring of both
MDCK epithelial monolayer and CG-ASCs in 3D microenvironment. The multiple Z-sections in confocal
images demonstrated higher columnar shapes in MDCKs when cocultured with CG-ASCs. Promotion of
cilia formation and functional expression of iron transport protein in MDCKs were also observed in the
current platform. Thus, this microfluidic coculture device provides renal epithelial cells with both
morphological and functional improvements that may avail to develop bioartificial renal chips.
Turing pattern formation without diffusion
Shigeru Kondo
Osaka University
The reaction-diffusion mechanism, presented by AM Turing more than 60 years ago, is currently the most
popular theoretical model explaining the biological pattern formation including the skin pattern. This theory
suggested an unexpected possibility that the skin pattern is a kind of stationary wave (Turing pattern or
reaction-diffusion pattern) made by the combination of reaction and diffusion. At first, biologists were quite
skeptical to this unusual idea. However, the accumulated simulation studies have proved that this
mechanism can not only produce various 2D skin patterns very similar to the real ones, but also predict
dynamic pattern change of skin pattern on the growing fish. Now the Turing’s theory is accepted as a
hopeful hypothesis, and experimental verification of it is awaited.
Using the pigmentation pattern of zebrafish as the experimental system, our group in Osaka University has
been studying the molecular basis of Turing pattern formation. We have identified the genes related to the
pigmentation, and visualized the interactions among the pigment cells. With these experimental data, it is
possible to answer the crucial question, “How is the Turing pattern formed in the real organism?”
The pigmentation pattern of zebrafish is mainly made by the mutual interactions between the two types of
pigment cells, melanophores and xanthophores. All of the interactions are transferred at the tip of the
dendrites of pigment cells. In spite of the expectation of many theoretical biologists, there is no diffusion of
the chemicals involved. However, we also found that the lengths of the dendrites are different among the
interactions, which can substitute the difference of diffusion constant in the RD model. Therefore the real
mechanism we found in the zebrafish skin is not the classic RD mechanism, but is mathematically
equivalent to the original Turing mechanism.
Collagen and Hyaluronan for Regenerative Medicine
Lynn LH Huang
National Cheng Kung University
Collagen and hyaluronan are important biomaterials as well as extracellular matrices of the bodies. Collagen
is a major scaffold as well as structural protein of connective tissues and possesses 25% to 35% of the
whole-body protein content. Hyaluronan plays key roles during embryonic developments and affects cell
migration, proliferation, differentiation, and tissue regeneration. Since 1990, we first proposed the function
of hyaluronan in conjunction with collagen gel and have series of studies and publications followed.
Recently we found that a seamless wound healing can be exerted from right combination of both. These
results provide new insights into the potential of artificial matrix in tissue engineering and imply good
potential for future improved applications in wound treatment.
Incorporation of stem cells with tissue engineering is a nowadays technology in regenerative medicine.
We further develop a series of novel tissue gels using collagen and hyaluronan for the purpose of stem cell
transplantation as well as tissue regeneration. We especially investigated the effects of hyaluronan on
mesenchymal stem cells because it is recognized as a critical component of the hematopoietic stem cell
niche and is widely distributed in mesenchymal tissues. Our previous finding suggests that hyaluronan
preserves the differentiation potential of long-term cultured murine adipose-derived stromal cells. We also
reported that hyaluronan substratum can hold human placenta-derived mesenchymal stem cells at a slow cell
cycling mode and with multidrug resistance phenotype, which are the characteristics of stemness.
Disrupted Ectodermal Organ Morphogenesis in Mice with a Conditional Histone Deacetylase 1, 2
Deletion in the Epidermis
Michael Hughes
University of Southern California
Histone deacetylases (HDACs) are present in the epidermal layer of the skin, outer root sheath, and hair
matrix. To investigate how histone acetylation affects skin morphogenesis and homeostasis, mice were
generated with a K14 promoter-mediated reduction of Hdac1 or Hdac2. The skin of HDAC1 null (K14-Cre
Hdac1cKO/cKO) mice exhibited a spectrum of lesions, including irregularly thickened interfollicular
epidermis, alopecia, hair follicle dystrophy, claw dystrophy, and abnormal pigmentation. Hairs are sparse,
short, and intermittently coiled. The distinct pelage hair types are lost. During the first hair cycle, hairs are
lost and replaced by dystrophic hair follicles with dilated infundibulae. The dystrophic hair follicle
epithelium is stratified and is positive for K14, involucrin, and TRP63, but negative for keratin 10. Some
dystrophic follicles are K15 positive, but mature hair fiber keratins are absent. The digits form extra
hyperpigmented claws on the lateral sides. Hyperpigmentation is observed in the interfollicular epithelium,
the tail, and the feet. Hdac1 and Hdac2 dual transgenic mice (K14-Cre Hdac1cKO/cKO Hdac2þ/cKO) have
similar but more obvious abnormalities. These results show that suppression of epidermal HDAC activity
leads to improper ectodermal organ morphogenesis and disrupted hair follicle regeneration and homeostasis,
as well as indirect effects on pigmentation.
The dedifferentiation and transdifferentiation of perivascular fibroblasts during digit tip regeneration
in mice
Ken Muneoka
Tulane University
The mouse digit tip shares with human fingertips the ability to regenerate following amputation injury.
This is one of a few models for epimorphic regenerative responses in adult mammals. During the
regeneration process a blastema of proliferating cells form and these cells redifferentiate into multiple cell
types, including bone, marrow, connective tissue and new vasculature. We have used a number of
approaches to investigate the cell source for the blastema and to address the plasticity of blastema cells. Our
studies demonstrate that perivascular fibroblastic cells of the bone marrow undergo a partial
dedifferentiation response to contribute to the blastema. These perivascular derived blastema cells display
multipotency
during
the
regeneration
by
re-differentiating
into
perivascular
fibroblasts
or
transdifferentiating into osteoblasts and endothelial cells. These studies are the first to identify a cell type in
mammals that possesses the ability to dedifferentiate and transdifferentiate during an epimorphic
regenerative response.
Regenerative Repair vs. Repair with Scars: Developmentally-Regulated Skin Wound Repair
Tai-Lan Tuan
University of Southern California
Scarring is a major medical problem that gives rise to adverse cosmetic, functional, and growth sequelae.
Prevention or reduction of scarring after surgery remains a major goal of surgeons. Early gestation fetal skin
heals in a regenerative fashion without scarring. An understanding of the mechanisms underlying scarless
fetal healing can provide answers that may be applicable to improving postnatal wound healing. We study if
the expression and function of the plasminogen activator/plasmin protease system, which is critical in blood
clot removal and tissue remodeling, plays a role in fetal skin wound repair. The results show that levels of
uPA (urokinase plasminogen activator) and PAI-1 (plasminogen activator inhibitor-1) are developmentally
regulated in fetal mouse skins. Of significance, the PAI-1 to uPA ratio increases when fetal skin wounds
transitioned from scarless (embryonic day [E]14) to scar-forming (E17 and after) repair. In addition, the
application of aprotinin, an inhibitor of uPA and plasmin, to skin wounds of E14.5 fetal mice causes
collagen scarring in normally scarless wounds. Finally, adenoviral overexpression and small interfering
RNA suppression demonstrate that PAI-1 produces elevated collagen accumulation in neonatal human
normal skin fibroblasts, supporting that PAI-1-targeted interventions may have therapeutic utility in the
prevention of scarring. (Supported by grants, R01GM095821 and R01GM055081, from NIGMS, NIH,
DHHS, USA)
Thrombomodulin regulates keratinocyte differentiation and promotes wound healing
Hua-Lin Wu
National Cheng Kung University
The membrane glycoprotein thrombomodulin (TM) has been implicated in keratinocyte differentiation and
wound healing, but its specific function remains undetermined. The epidermis-specific TM knockout mice
were generated to investigate the function of TM in these biological processes. Primary cultured
keratinocytes obtained from TM(lox/lox); K5-Cre mice, in which TM expression was abrogated, underwent
abnormal differentiation in response to calcium induction. Poor epidermal differentiation, as evidenced by
downregulation of the terminal differentiation markers loricrin and filaggrin, was observed in TM(lox/lox);
K5-Cre mice. Silencing TM expression in human epithelial cells impaired calcium-induced extracellular
signal-regulated kinase pathway activation and subsequent keratinocyte differentiation. Compared with
wild-type mice, the cell spreading area and wound closure rate were lower in keratinocytes from
TM(lox/lox); K5-Cre mice. In addition, the lower density of neovascularization and smaller area of
hyperproliferative epithelium contributed to slower wound healing in TM(lox/lox); K5-Cre mice than in
wild-type mice. Local administration of recombinant TM (rTM) accelerated healing rates in the TM-null
skin. These data suggest that TM has a critical role in skin differentiation and wound healing. Furthermore,
rTM may hold therapeutic potential for the treatment of nonhealing chronic wounds.
Topology Dependent Hair Plucking Leads to Non-Linear Amplification of Regenerative Response via
a Distress-Gauging Cellular Circuit
Chih-Chiang Chen
Department of Dermatology in Taipei Veterans General Hospital
Recent works showed that activation of hair stem cells is controlled by both intra- and extra-follicular
factors. With this premise, we hypothesize that single intra-follicular hair plucking might elicit signaling
changes that can trigger the regeneration of neighboring unplucked follicles. Here we show, with proper
spacing, we can activate 1000 hairs to regenerate from telogen (resting stage) even only 200 hairs were
plucked. Gene profiling, in situ expression, bead-mediated protein delivery and genetic mutant analyses
identify
a
multi-
follicle
anagen re-entry (both plucked and unplucked follicles). We simulate the cellular circuit behavior based on a
recent mathematical model treating the growth dynamics of hair follicle stem cell populations as a stochastic
excitable medium. The results demonstrate that a generic distress-gauging cellular circuit can integrate
existing cell injury, immune and regeneration related molecular pathways from multi-organs to quantify the
distress and decide to disregard it as negligible or respond with a full scale non-linear amplified response
when a threshold is reached.
The regeneration of complex structures from axolotls to mice: the evolution
of the
blastema and
its stem cells.
Malcolm Maden PhD
University of Florida
Axolotls are the champions of regeneration and have provided us with a wealth of data on the regeneration
of complex structures such as the limb.
After limb amputation, wound healing generates a signaling
epidermis, the apical epidermis, which is crucial to the development and proliferation of the blastema cells.
We have recently performed a microarray experiment to identify the signaling properties of the apical
epidermis and found five developmental signaling pathways that are limb specific and not up-regulated
during normal flank wound healing. This allows a blastema to form on the regenerating limb which consists
of a heterogeneous group of lineage restricted cells. We consider these two events crucial to regeneration
and have asked whether they also occur in mammals as a prelude to complex regenerative events.
The
African spiny mouse can regenerate tissues after a 4mm punch through the ear – cartilage, skin, hairs dermis
– and in this case we believe that the equivalent of the axolotl apical epidermis and the equivalent of a
blastema forms during this regenerative process which provides the key to the successful regeneration in
spiny mice. We have also compared flank wound healing in axolotls, spiny mice and the lab mouse.
Axolotls and spiny mice can heal wounds completely scar-free manner and show many similar
characteristics particularly in the extracellular matrix of the wound whereas the lab mouse heals with a scar
and has a different composition to the matrix. These studies suggest that the requirements for successful
regeneration of complex structures may be common between regenerating species throughout the
vertebrates.
Mechanics-driven Self-organization in Tissue-scale Tubulogenesis Biography
Chin-Lin Guo
California Institute of Technology, Bioengineering.
The ability of cells to self-organize with extracellular matrix (ECM) molecules into tissue-scale structures
raises the possibility that an understanding of such processes can lead to a scaffold-free approach for tissue
engineering. While the signaling cascades in cell-ECM interactions are extensively studied, how the
dimensionality in cell-ECM and cell-microenvironment interactions leads to various morphogenetic
outcomes is not well understood. Here, we show three distinct phenotypes. First, when epithelial cells are
surrounded by 3-D ECM, they can develop long-range mechanical interactions (up to 600 micrometers).
Second, when these cells are cultured on 2-D ECM and interacting with collagen fibers in the media, they
can self-organize into branched tubular structures (up to millimeter-long, tens of micrometer-wide) by
collective migration. Third, when cells are cultured in suspension (as the early stage of embryo) and
interacting with collagen fibers in the media, they can form unbranched, centimeter-long, hundreds of
micrometer-wide tubules with highly organized architectures. In particular, while the first phenotype
depends on cell types and might require specific morphogens, the second and the third phenotypes are
observed in several epithelial cell lines and do not need specific morphogens. Moreover, the first phenotype
is observed in many tumor cell lines and can advance their invasion, whereas the other two phenotypes are
not found in these cells. Our results provide a novel framework for scaffold-free, tissue-scale engineering
and a quantitative platform for investigating the phenotypic differences between normal and tumor cells.
Defining the stromal niche for stem cells of specialized epidermis
John Foley
Indiana University School of Medicine
There have been tremendous advances in understanding the role of various signaling pathways in the control
of the hair follicle stem cell niche in dorsal skin of the trunk. This skin plays a largely protective role, but
mammals interface and manipulate their environment through small patches of specialized epidermis. These
tend to be glabrous and in the human include lips, palms, soles anal/genital regions and the nipple/areola.
The unique characteristics of these sites are thought to be result from modulations of the basic
epidermal-mesenchymal interactions mediated by unique regional fibroblast populations. Little is known
about the functional role of other cell types present in these stromal niches. Most examples of specialized
epidermis change very little throughout life, but the human and mouse nipple/areola expands under the
influence of pregnancy and lactation hormones, and we are leveraging this biology to ascertain the
relationship between fibroblasts and muscle cell types and determine how this influences epidermal stem
cells. To this end we have characterized the epidermal, connective tissue and hair follicle changes that
accompany expansion of the nipple/areola during pregnancy and lactation. We have established a role for
both the exposure to pregnancy and lactation hormones as well as mechanical strain in this growth. Using
a novel bitransgenic mouse, we have sorted large populations of nipple connective tissue fibroblasts and
smooth muscle cells. We have established their ability to induce nipple-like skin in grafts, and probed the
hormonal sensitivity of these two cell types.
Finally, we have begun establishing the specific gene
expression profile for nipple connective tissue niche.
The Regeneration of Feathers after different levels of wounding
Ting-Xin Jiang, Ping Wu, Ang Li, Randall B Wideliz, Cheng-Ming Chuong
Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles,
California, USA.
One main unsolved challenge in regenerative medicine is how to elicit and harness the power of
regeneration. Following injury, human tissues mostly respond with repair, covering the wound with
epidermis and connective tissue without restoring complete function 1. In contrast, many animals regenerate
whole organs
2
with mechanisms distilled through evolution, and can inspire us to envision new enhanced
regeneration strategies. Here we explore the feather model which shows robust regenerative power and a
distinct stem cell niche architecture 3. During feather cycling, feather stem cells assume a ring-configuration,
ascending and descending cyclically within the proximal follicle 4. We challenge the feather’s regeneration
potential upon the loss of its stem cells. First we deplete stem cells by plucking growth phase feathers.
Surprisingly, feathers can still regenerate. Cell tracking shows the remaining epithelia in the lower follicle
sheath are now re-directed to re-epithelialize the denuded dermal papilla. The new blastema regenerates
collar bulge stem cells and regrows the feather. Latent stem cell potential in the plucking-activated follicle
sheath is evaluated by chicken/quail transplantation. Activation is accompanied by K15, Lgr6, Sox 9 and
nuclear -catenin expression. In resting phase, the follicle base can be removed together with papillar
ectoderm stem cells and dermal papilla. No feathers can regenerate without the dermal papilla. With a
transplanted dermal papilla, host cells mix with donor cells to re-establish a new regenerative wound field,
capable of generating different numbers and sizes of feathers. The work suggests that bulge stem cells are
not a fixed entity but represent a functional state. This extra-ordinarily large progenitor pool, capable of
quickly adapting to changes to re-establish stem and TA hierarchy, endows feathers with robust regenerative
power. This insight can help us develop new strategies for regenerative medicine.
Abstract for poster section
大會展示海報摘要
編號:P1
Recombinant Lectin-like Domain of Thrombomodulin Suppresses Vascular Inflammation by
Reducing Leukocyte Recruitment via Interacting with Lewis Y on Endothelial Cells
Wei-Ling Lin1,2,3; Chuan-Fa Chang5; Chung-Sheng Shi1; Guey-Yueh Shi1,3*; and Hua-Lin Wu1,3,4*
1
From Department of Biochemistry and Molecular Biology, 2Institute of Basic Medical Sciences, 3Cardiovascular
Research Center, 4Center of Bioscience and Biotechnology, 5Department of Medical Laboratory Science and
Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
The N-terminal lectin-like domain (domain 1 [D1]) of thrombomodulin (TM) is known to have an
anti-inflammatory function. We previously showed that recombinant TM domain 1 (rTMD1) interacts with a
carbohydrate molecule, Lewis Y (Ley), which is found to be expressed on adhesion molecules and involved
in cell adhesion. Here, we tested the effect of rTMD1–Ley interaction on leukocyte recruitment in
inflammation. The expression of Ley on the surface of human umbilical vein endothelial cells was increased
by tumor necrosis factor-α stimulation. Direct binding of rTMD1 to Ley on the cell surface was observed.
rTMD1 inhibited Ley-mediated leukocyte adhesion on the Ley-immobilized flow chamber and activated
endothelium under a shear flow. The following leukocyte transmigration to endothelium was also reduced
by rTMD1 through binding Ley. In vivo, treatment of rTMD1 reduced leukocyte recruitment to the
inflammatory sites in carotid ligation injury and thioglycollate induced peritonitis. rTMD1 administration in
apolipoprotein E–deficient mice effectively suppressed atherosclerotic plaque formation and macrophage
infiltration in atherosclerotic lesions. Increased Ley expression, as well as administered rTMD1, was
observed in inflamed vessels. rTMD1 suppresses vascular inflammation by inhibiting leukocyte recruitment
to endothelium through attenuating Ley-mediated adhesion and further protects against atherosclerosis
progression. The present study provides a mechanism showing that rTMD1 can inhibit inflammation by
binding to its carbohydrate ligand Ley.
編號:P2
Functional recoveries of sciatic nerve regeneration by combining chitosan-coated conduit and
neurosphere cells induced from adipose-derived stem cells
Yuan-Yu Hsueh, Ya-Ju Chang, Tzu-Chieh Huang, Shih-Chen Fan, Duo-Hsiang Wang, Jason J.J. Chen,
Chia-Ching Wu*, Sheng-Che Lin*
Suboptimal repair occurs in a peripheral nerve gap, which can be partially restored by bridging the gap with
various biosynthetic conduits or cell-based therapy. In this study, we developed a combination of chitosan
coating approach to induce neurosphere cells from human adipose-derived stem cells (ASCs) on
chitosan-coated plate and then applied these cells to the interior of a chitosan-coated silicone tube to bridge a
10-mm gap in a rat sciatic nerve. Myelin sheath degeneration and glial scar formation were discovered in the
nerve bridged by the silicone conduit. By using a single treatment of chitosan-coated conduit or neurosphere
cell therapy, the nerve gap was partially recovered after 6 weeks of surgery. Substantial improvements in
nerve regeneration were achieved by combining neurosphere cells and chitosan-coated conduit based on the
increase of myelinated axons density and myelin thickness, gastrocnemius muscle weight and muscle fiber
diameter, and step and stride lengths from gait analysis. High expressions of interleukin-1beta and
leukotriene B4 receptor 1 in the intra-neural scarring caused by using silicone conduits revealed that the
inflammatory mechanism can be inhibited when the conduit is coated with chitosan. This study
demonstrated that the chitosan-coated surface performs multiple functions that can be used to induce
neurosphere cells from ASCs and to facilitate nerve regeneration in combination with a cells-assisted coated
conduit.
編號:P3
Identifying the Spatial Generation of Distinct Filament Elasticity in a Single Cell: May the Force Lead
the Way
Hans I-Chen Harn1, Yang-Kao Wang2, Yi-Wei Huang3, Wen-Tai Chiu1,4, Hsi-Hui Lin3, Chao-Min Cheng5,
Ming-Jer Tang1,3
1
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan;
2
Graduate Institute of Biomedical Materials and Tissue Engineering, School of Oral Medicine, Taipei Medical
University, Taipei 110, Taiwan; 3Department of Physiology, College of Medicine, National Cheng Kung University,
Tainan 701, Taiwan; 4Department of Biomedical Engineering, College of Engineering, National Cheng Kung
University, Tainan 701, Taiwan; and 5Institute of Nanoengineering and Microsystems, National Tsing Hua University,
Hsinchu 300, Taiwan
Any cellular response leading to morphological changes is highly tuned to balance the force generated from
structural reorganization, provided by actin cytoskeleton. Actin filaments also serve as the backbone of
intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. We
proposed the mechanical and spatial organization of actin filaments reflect the status quo of a cell. Here we
explored the mechanical properties and the distribution of actin filaments in single living cells using a
Bio-AFM, and determined their contributions to the mechanical properties and the behavior of the cell. A
living fibroblast was found to display filament elasticity at around 35 kPa, and both low substrate rigidity
and various cytoskeleton-related inhibitors dose dependently decreased filament and cell elasticity. In both
3T3 fibroblasts and U2OS osteosarcoma cells, filament elasticity were uniform in the static and rounded
state, while in mobile and polarized state the filaments elasticity were higher in the leading head and softer
in the rear tail. When U2OS cells were seeded on soft substrate or treated with actin cytoskeleton-related
inhibitors, the percentage of polarized cells was vastly reduced, signifying the importance of generating
distinct filament elasticity. To delineate the role of force in regulating filament elasticity and cell behavior,
we used microfabricated post-array detectors to show that a static and rounded fibroblast generated greater
total force but the net vector force was closer to zero than that of a mobile and polarized one. Furthermore,
the forces generated on the leading head of a polarized fibroblast were greater than those of its rear tail. This
study connects the spatial and mechanical properties of actin filaments to the overall elasticity and behavior
of a living cell, and highlights the contribution of force generation of actin filaments in leading cell
polarization during directional migration.
編號:P4
Cell therapy for hypoxic-ischemic brain injury by neural and endothelial progenitor cells derived
from adipose-derived stem cells
Ya-Ju Chang1,2, Yuan-Yu Hsueh2, 3, 4, Chia-Wei Huang1, Yi-Lun Chiang1,2, Fitri Handayani2,
Chao-Ching Huang3, 5, Sheng-Che Lin4, Chia-Ching Wu1, 2, 6
1
Institute of Basic Medical Sciences, National Cheng Kung University.2 Department of Cell Biology and Anatomy,
National Cheng Kung University.3 Institute of Clinical Medicine, National Cheng Kung University. 4 Division of
Plastic Surgery, National Cheng Kung University Hospital. 5 Department of Pediatrics, National Cheng Kung
University Hospital. 6 Department of Biomedical Engineering, National Cheng Kung University
Adipose-derived stem cells (ASCs), isolated from adipose tissue, are easily and abundantly harvested than
other tissues. ASCs could differentiate into cardiomyocyte, myocyte, chondrocyte, and osteocyte under
different stimulations by using growth factors. The plasticity of ASCs would be applied to regenerative
medicine or cell-based therapy. The purpose of this study is testing the benefit of cell therapy in neonatal
hypoxic-ischemic (HI) brain injury by using neural progenitor cells (NPCs) and/or endothelial progenitor
cells (EPCs) derived from ASCs. Recently, we demonstrated that ASCs has the possibility to differentiate
toward NPCs and EPCs. The ASCs formed sphere-like structure when seeding on chitosan coated surface
for 72 hours. In NPCs differentiation, the sphere expressed NPCs markers in both protein and gene levels for
nestin, neurofilament heavy chain (NFH), and glial fibrillary acidic protein (GFAP). The combination of
chemical growth factor and mechanical shear stress facilitated EPCs differentiation to induce ASCs express
the EPCs markers. The endothelial functions were demonstrated in the tube formation and DiI-LDL
uptaking for these differentiated EPCs. The HI brain injury was created by unilateral common carotid artery
ligation and then subjected to 8% oxygen hypoxia for 2 hours in the postnatal day 7 SD rat pups. The NPCs
and/or EPCs were transplanted to the HI injured rats by intraperitoneal injection. Lower brain loss area and
cell apoptosis were observed by Nissl staining and TUNEL staining in the HI brain for animals received
various cell therapies for 7 days. The immunofluorescence staining of nestin, NFH, GFAP and RECA
proved the preservation of both neurological and endothelial structures on injured brain after cell
transplantation. Tracing the transplanted cells discovered the enhancement of neurogenesis and angiogenesis
by integrating these cells into the neurovascular units. Therefore, the NPCs and/or EPCs derived from ASCs
can be a potential cell source for cell therapy in HI brain injury.
編號:P5
Keloid fibroblasts exhibit hyper-responsiveness to mechanical stimulation: the role of cell softening
and nuclear Runx2
Chao-Kai Hsu1,2, Hsi-Hui Lin3, Yang-Kao Wang4, Hans I-Chen Harn3, Chao-Min Cheng5, Shyh-Jou Shieh6,
Julia Yu-Yun Lee1, and Ming-Jer Tang3
1
Institute of Clinical Medicine, 3Department of Dermatology2, Department of Physiology, 4Department of Medicine,
Skeleton-Joint Research Center, 6Department of Surgery, National Cheng-Kung University College of Medicine and
Hospital, Tainan, Taiwan; Institute of Nanoengineering and Microsystems5, National Tsing Hua University, Hsinchu,
Taiwan
Keloids are pathological scars characterized by excessive extracelluar matrix (ECM) production and prone
to form in the body area with increased skin tension. We hypothesize that keloid results from the
hyper-responsiveness of keloid fibroblasts (KFs) to mechanical stimulation. The purpose of the study is to
understand the mechanical properties of KFs and the role of nuclear Runx2, an osteogenic and chondrogenic
transcription factor, in their responsiveness to mechanical stimulation. By using atomic force microscopy,
we found that KFs (1,205±70 pascal) tended to be softer than normal fibroblasts (NFs) (1,521±145 pascal)
(N=7, P=0.0744), while keloid tissue and normal skin tissue were measured at 16,570±1,648 and 1,503±250
pascal, respectively (N=3, P≦0.001). We plated KFs and NFs on collagen-coated polyacrylamide gel of
different stiffness simulating tissue microenvironment, and found that KFs produced more fibronectin than
NF when cultured on a stiff gel (20,000 pascal) while both KFs and NFs produced equally low level of
fibronectin on a soft gel (2,000 pascal). Tissue protein analysis revealed that keloid tissues were abundant
with fibronectin, type 1A1 collagen, type 3A1 collagen and skeleton-associated protein collagen type 11A1.
From our immunohistochemical study, the upstream transcription factor, Runx2, was detected in the nuclei
of fibroblasts within keloid lesions. Knocking down Runx2 in KFs using RNA interference decreased the
ECM production, including fibronectin, type 3A1 collagen and type 11A1 collagen. On polyacrylamide gel
culture system, the nuclear expression of Runx2 in KFs correlated with substratum stiffness. In conclusion,
KFs and NFs display different profile of biomechanical properties and response to changes in substratum
stiffness. Ectopic expression of Runx2 possibly plays a role in the hyper-responsiveness of keloid to
mechanical stimulation.
編號:P6
Non-thermal micro-plasma exposure for healing burn wound on mice
Minh-Hien Ngo, Pei-Lin Shao, Jiunn-Der Liao*
Department of Materials Science and Engineering, National Cheng Kung University
Healing burn wound research has received attractions in recent decades, especially the second degree burn,
which may reach the epidermis and partial dermis layers. Several methods and techniques have been applied
to manage the burn injuries, such as different kinds of dressings, pharmacotherapies and plasma treatment [1].
The latter has been increasingly studied. In this work, non-thermal N2/Ar micro-plasma was applied to
enhance healing on the second degree burn wound mice through the wound area reduction. Six wounds were
created in the dorsal of each mouse (6 mice in total) by solid aluminum bar with 5 mm in diameter (46 g)
and an average temperature of 70 ± 3oC. In Figure 1(a) and (b), the parameters for burn wound exposure at
13 W and 0.5% N2 addition were chosen, in view of maintaining plasma plume temperature < 40°C
(appropriate to the body temperature in mouse) and corresponding with relatively high NO peak intensity.
N2/Ar micro-plasma was utilized to expose upon the burn wound achieved mice in three groups: (1)
immediately after the burn achievement (P1), (2) continuous exposure until post-burn day 2 (P3), and (3)
continuous exposure until post-burn day 4 (P5). Dressing and gas flow exposure were also conducted as well
for the references. The burn wounds were assessed every day to examine the wound size. Until post-burn
day 18, the mice were sacrificed for H&E staining. Figure 1(c) showed that the wound area reduction rate
was higher for the cases of N2/Ar micro-plasma exposed wounds than those of gas flow exposed and
dressing ones, while the control group exhibited the lowest wound area reduction rate. From our preliminary
study, non-thermal N2/Ar micro-plasma is presumably effective for the stimulation of healing burn wound
on mice.
編號:P7
CCN1/α6β1 Mediates Myocardial Injuries Induced by Work Overload or by Doxorubicin in Mice
Pei-Ling Hsu1,2, Bor-Chyuan Su1,2, and Fan-E Mo1,2
1
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan and
2
Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, 1 University Road,
Tainan 701, Taiwan
Matricellular protein CCN1 is expressed in myocardial infarction, pressure overload, and ischemia in mice,
and in patients with a failing heart. Despite its well-documented angiogenic activities, CCN1 promotes
fibroblast apoptosis in some contexts. The role of CCN1 in an injured heart was not clear. We assessed the
hypothesis that CCN1 plays a detrimental role and mediates cardiac injury through its proapoptotic activities.
To test the role of CCN1 in cardiac injury, we employed two different myocardial injury models in mice,
including a work-overload-induced injury created by isoproterenol treatment (ISO; 100 mg/kg/day; s.c. for 5
days; n= 6 for each group) and an injury induced by the cardiotoxicity of doxorubicin (DOX, single dose of
15 mg/kg; i.p. sacrificed after 14 days). Ccn1 expression was induced in the damaged myocardium in both
injury models. A line of knock-in mice carrying an apoptosis-defective Ccn1 mutant allele, Ccn1-dm, which
has disrupted integrin
6
1
binding sites, were tested in the ISO- or DOX -induced cardiac injury.
Myocardial damage was seen in tissues from wile-type (WT) hearts after receiving ISO.
Ccn1dm/dm (DM)
mice possessed remarkable resistance against ISO or DOX treatments and exhibited no tissue damage or
fibrosis compared to WT mice after H&E or Masson’s trichrome stainings. DM mice were resistant to both
ISO- and DOX-induced cardiac cell apoptosis, indicating that CCN1 is critically mediating cardiomyocyte
apoptotic death in cardiac injury. Moreover, we found that death factor Fas ligand (FasL) and its receptor
Fas were upregulated in WT mice receiving ISO or DOX treatments by immunohistochemical staining,
compared with the PBS-control. 8-OHdG-positive, a marker for oxidative stress, cardiomyocytes were
increased by ISO or DOX treatments as well. In contrast, the expression of Fas/FasL, and the
8-OHdG-positive cardiomyocytes in the myocardium of DM mice were not changed by ISO or DOX. We
identify CCN1 as a novel pathophysiological regulator of cardiomyocyte apoptosis in cardiac injury.
Blocking apoptotic function of CCN1 effectively prevents myocardial injury in mice. CCN1 and its receptor
6
1
represent promising future therapeutic targets in cardiac injury.
編號:P8
Efficacy study of non-thermal micro-plasma for promoting CO2 laser irradiation wound healing
Pei-Lin Shao, Yi-Cheng Wang, Minh-Hien Ngo, Yu-Shiang Liu, Jiunn-Der Liao*
Department of Materials Science and Engineering, National Cheng Kung University
Advanced wound management is one of the major public health concerns. The laser irradiation is the
frequently used method for the facial problems such as wrinkle, scar. The common clinical care of the
laser-irradiated wound has been taken by healing ointment, but still feel uncomfortable, tingling and allergy
problem for some patients [1]. Recently, plasma therapy has attracted widespread interests which provide
advantages such as contact-free, painless, non-allergy, and with antibacterial effect for wound management.
In this study, non-thermal micro-plasma technique was introduced, which was generated by radio frequency.
The working distance between skin surface and micro-plasma was ca. 4 mm. The optimized micro-plasma
plume temperature was controlled ca. 37°C by adjusting the applied power and N2 in Ar. Plasma species
was measured using an optical emission spectroscopy, while NO, OH, O, and Ar were the major species in
this micro-plasma system. In the in vivo studies, animal modeling assessment was divided into three
categories: non-treatment (NT), one-time plasma treatment (PT1), and three-time plasma treatment (PT3).
Our results show that the reduction percentage of wound area at the 21 postoperative days (POD) was 80%,
90% and 100% for NT, PT1 and PT3, respectively. The remodeling of dermal-epidermal junction,
re-epithelialization, and well-developed granulation tissues were observed at 7 POD for PT1 and PT3 groups
and 14 POD for NT group. The quantification of OCT image intensity at 14 POD and blood flow intensity at
21 POD was measured by comparing with that at 1 POD. The results suggest that for NT, PT1, PT3 groups,
the increases of 49.6, 87.6, and 87.2% (14 POD) and 37.0, 35.2, and 60.4% (21 POD) were obtained. In
summary, this novel non-thermal micro-plasma technique is promising to be employed as an effective
treatment for healing and remodeling the laser-irradiated wound in the dermal-epidermal junction.
編號:P9
CCN1 enables Fas ligand-induced apoptosis by elevating Fas expression in primary cardiomyocytes
or by increasing cytoplasmic Smac in cardiomyoblast H9c2 cells
Bor-Chyuan Su1,2, Pei-Ling Hsu1,2, Fan-E Mo1,2
1
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan and
2
Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, 1 University Road,
Tainan 701, Taiwan
Fas/Fas ligand (FasL) is implicated in cardiac ischemia/reperfusion injury. However, cardiomyocytes in
culture are resistant to FasL-induced apoptosis, suggesting that additional factor(s) are required for
FasL-induced apoptosis. Matricellular protein CCN1/CYR61 has been demonstrated to promote cytotoxicity
of FasL in human skin fibroblasts. CCN1 is induced in a variety of cardiac pathologies. We assessed the
hypothesis that CCN1 may be involved in the regulation of FasL-induced apoptosis in cardiomyocytes. We
found that either FasL or CCN1 did not induce cell death in neonatal rat ventricular cardiomyocytes
(NRVM). Interestingly, the combination of FasL+CCN1 induced 2-fold induction of apoptosis (p<0.001).
An integrin-α6β1-binding defective mutant CCN1, CCN1-DM failed to exert synergy with FasL to induce
apoptosis, indicating that CCN1 promotes cardiomyocyte apoptosis through its engagement with the integrin
α6β1. The engagement between CCN1 and α6β1 instigated the elevation of cellular reactive oxygen species
(ROS), the activation of mitogen activated protein kinase (MAPK) p38, and followed by the induction of
cell surface display of Fas, thereby sensitizing NRVM to FasL-induced apoptosis. Pretreatment of the
MAPK p38 inhibitor SB202190 abolished the CCN1-induced cell-surface Fas expression and the apoptosis
induced by FasL+CCN1. In addition, we found that FasL or CCN1 alone did not cause apoptosis in H9c2
cardiomyoblasts, and required the combination of FasL+CCN1 to induced apoptosis (p<0.001) reminiscent
of observed in NRVM.
Mechanistically, CCN1 acted through binding to integrin α6β1, ROS generation,
and MAPK p38 activation. However, CCN1 did not increase the expression of cell surface Fas for its
synergy with FasL in H9c2 cells. Instead, CCN1 induced Bax translocation to mitochondria, which in turn
led to the release of Smac from mitochondria to cytosol. The cytosolic Smac functions to neutralize XIAP.
Smac is critical for CCN1 action, because the knockdown of Smac blunted the apoptotic activities of CCN1.
In conclusion, CCN1 may play a detrimental role in a stressed heart to both the differentiated
cardiomyocytes and the proliferative cardioblasts through distinct signaling mechanisms.
編號:P9
Mechanobiology of epithelial-mesenchymal-transition in breast cancer cell line: role of vimentin
Ching-Yi Liu1, Yang-Kao Wang2, Hsi-Hui Lin1, Hans I-Chen Harn1 and Ming-Jer Tang1
1Department of Physiology, National Cheng-Kung University, Tainan,2Graduate Institute of Biomedical Materials and
Tissue Engineering, Taipei Medical University, Taiwan
Disruption of actin filaments and the decrease in focal adhesions are common features of transformed cells
displaying anchorage-independent growth and cellular tumorigenicity. However, some cancer cells undergo
epithelial-mesenchymal-transition (EMT) and become more malignant, a process accompanied by the lost of
cell-cell junction and gain of mesenchymal markers such as vimentin. In this study, we hypothesized that
intermediate filament vimentin may function as a force transmission linker between actin filament and
microtubules in EMT cancer cells. To test this hypothesis, we first evaluated the biophysical properties of
three breast cancer cell lines, MCF7, MDA-MB 468, and MDA-MB 231 by employing atomic force
microscopy (AFM) to measure cell stiffness. The results revealed that among these breast cancer cell lines,
MDA-MB 231 showed the highest cell stiffness. Western blot analysis showed that MCF7 expressed the
lowest levels of β1 integrin and FAK, whereas MDA-MB 231 displayed the highest levels of EMT-related
proteins, such as 1 integrin, FAK, vimentin, and fibronectin. With the vimentin si-RNA application in
MDA-MB 231 cells, these cells become larger and the cell stiffness was decreased. The
immunofluorescence staining also showed that actin filament and microtubule were reorganized toward cell
edge when vimentin knockdown. Further, the AFM indentation revealed that the reorganized cytoskeletons
increased cell stiffness at cell edge but decreased the nucleus stiffness. Taken together, our studies showed
that vimentin may not only function as a cell tension transmitter, but also a regulator for the maintenance of
cytoskeleton architecture and the balance of cell force generation.