A PROJECT REPORT

ON

Effect of Alcohol on Growth, Cytoskeletal Organization

And
Differentiation of Human Mesenchymal Stem cells

Submitted by

ABHIYAN VIPLAV

In the partial fulfillment for award of the degree

Of

Bachelor of Technology

Amity University, Noid

 Abstract

Mesenchymal stem cells (MSCs) represent a class of multipotent progenitor cells that have been
isolated from multiple tissue sites. Human bone marrow mesenchymal stem cells (hMSC) in vivo
differentiate into several phenotypes including osteogenic and adipogenic cells, both of which
remain as resident populations of bone marrow. From the previous studies it has been shown that
Alcohol has been associated with osteoporosis and osteonecrosis in patients and in animal
models. The decrease in bone volume associated with osteoporosis and age-related osteopenia is
accompanied by increased marrow adipose tissue formation. Recent studies have demonstrated
that alcohol contributes to abnormal lipid metabolism in the stem cells of bone marrow, but the
mechanisms have not been defined. Reversal of this process may provide a novel therapeutic
approach for osteopenic disorders. The purpose of this study is to examine the effect of alcohol
(EtOH) in the growth, cytoskeleton and the adipogenesis of the mesenchymal stem cells derived
from human bone marrow. Here, we cultured MSCs in three different conditions and examined
that alcohol in the medium acts as an inducing agent for adipogenesis during the process of
differentiation.
Introduction

HeLa cells are the classic example of an immortalized cell line. These are human epithelial cells
from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18). (Henrietta
Lacks, 1951) HeLa cells are termed "immortal" in that they can divide an unlimited number of
times in a laboratory cell culture plate as long as fundamental cell survival conditions are met.
The ability of these cells to survive is attributed to the active version of the enzyme called
"telomerase". The role of telomerases is to add certain DNA repeat sequences "TTAGGG" at the
end of chromosomes called" telomeres" and hence providing stability to chromosomes.
However, as the cell divide these telomeres shorten at each DNA replication and eventually will
no longer be able to divide. However, cancer or immortal cells have an active version of this
enzyme and hence preventing it from dying. This special characteristic of the Hela allows it to
divide unlimited number of times without loss of genomic instability. There are many strains of
HeLa cells as they continue to evolve by being grown in cell cultures, but all HeLa cells are
descended from the same tumor cells removed from Mrs Lacks. HeLa cells circumvent the
Hayflick Limit, which is the limited number of cell divisions that most normal cells can undergo
before dying in cell culture. It has been estimated that the total number of HeLa cells that have
been propagated in cell culture far exceeds the total number of cells that were actually in
Henrietta Lacks' body. Because of their avid adaptation to growth in tissue culture plates, HeLa
cells are sometimes difficult to control.

Stem cells

Stem cells have the remarkable potential to develop into many different cell types in the body
during early life and growth. In addition, in many tissues they serve as a sort of internal repair
system, dividing essentially without limit to replenish other cells as long as the person or animal
is still alive. When a stem cell divides, each new cell has the potential either to remain a stem
cell or become another type of cell with a more specialized function, such as a muscle cell, a red
blood cell, or a brain cell. Stem cells are distinguished from other cell types by two important
characteristics.
 1. They are unspecialized cells capable of renewing themselves through cell division,
sometimes after long periods of inactivity.
2. Under certain physiologic or experimental conditions, they can be induced to become
tissue- or organ-specific cells with special functions.

Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo,
called a blastocyst, the inner cells give rise to the entire body of the organism, including all of the
many specialized cell types and organs such as the heart, lung, skin, sperm, eggs and other
tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of
adult stem cells generate replacements for cells that are lost through normal wear and tear,
injury, or disease.Given their unique regenerative abilities, stem cells offer new potentials for
treating diseases such as diabetes, and heart disease. However, much work remains to be done in
the laboratory and the clinic to understand how to use these cells for cell-based therapies to treat
disease, which is also referred to as regenerative or reparative medicine.

Research on stem cells continues to advance knowledge about how an organism develops from a
single cell and how healthy cells replace damaged cells in adult organisms. Stem cell research is
one of the most fascinating areas of contemporary biology, but, as with many expanding fields of
scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates new
discoveries
 Review of Literature

Until recently, scientists primarily worked with two kinds of stem cells from animals and
humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells. The functions
and characteristics of these cells will be explained in this document. Scientists discovered ways
to derive embryonic stem cells from early mouse embryos nearly 30 years ago, in 1981. The
detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to
derive stem cells from human embryos and grow the cells in the laboratory. These cells are
called human embryonic stem cells. The embryos used in these studies were created for
reproductive purposes through in vitrofertilization procedures. When they were no longer needed
for that purpose, they were donated for research with the informed consent of the donor. In 2006,
researchers made another breakthrough by identifying conditions that would allow some
specialized adult cells to be "reprogrammed" genetically to assume a stem cell-like state. This
new type of stem cell, called induced pluripotent stem cells (iPSCs), will be discussed in a later
section of this document.

Stem cells of two types-

1. Embryonic stem cells are derived from embryos. Most embryonic stem cells are derived
from embryos that develop from eggs that have been fertilized in vitro—in an in vitro
fertilization clinic—and then donated for research purposes with informed consent of the
donors. They are not derived from eggs fertilized in a woman's body. Embryonic stem
cells (ESCs) provide an unlimited supply of progenitor cells for tissue engineering.
However, successful use of ESCs for clinical application requires the development of
efficient methods to differentiate cells into specific lineages in vitro and reconstituting
tissue function in vivo.

2. Adult stem cell is thought to be an undifferentiated cell, found among differentiated cells
in a tissue or organ that can renew itself and can differentiate to yield some or all of the
major specialized cell types of the tissue or organ. The primary roles of adult stem cells
in a living organism are to maintain and repair the tissue in which they are found.
Human embryonic and adult stem cells each have advantages and disadvantages regarding
potential use for cell-based regenerative therapies. One major difference between adult and
embryonic stem cells is their different abilities in the number and type of differentiated cell types
they can become. Embryonic stem cells can become all cell types of the body because they are
pluripotent. Adult stem cells are thought to be limited to differentiating into different cell types
of their tissue of origin. Embryonic stem cells can be grown relatively easily in culture. Adult
stem cells are rare in mature tissues, so isolating these cells from an adult tissue is challenging,
and methods to expand their numbers in cell culture have not yet been worked out.

Embryonic stem cells can be grown relatively easily in culture. Adult stem cells are rare in
mature tissues, so isolating these cells from an adult tissue is challenging, and methods to expand
their numbers in cell culture have not yet been worked out. This is an important distinction, as
large numbers of cells are needed for stem cell replacement therapies.
Scientists believe that tissues derived from embryonic and adult stem cells may differ in the
likelihood of being rejected after transplantation. We don't yet know whether tissues derived
from embryonic stem cells would cause transplant rejection, since the first phase 1 clinical
trial testing the safety of cells derived from hESCS has only recently been approved by the
United States Food and Drug Administration (FDA).

Adult stem cells, and tissues derived from them, are currently believed less likely to initiate
rejection after transplantation. This is because a patient's own cells could be expanded in culture,
coaxed into assuming a specific cell type (differentiation), and then reintroduced into the patient.
The use of adult stem cells and tissues derived from the patient's own adult stem cells would
mean that the cells are less likely to be rejected by the immune system. This represents a
significant advantage, as immune rejection can be circumvented only by continuous
administration of immunosuppressive drugs, and the drugs themselves may cause deleterious
side effects

Adult Mesenchymal stem cells: Characterization and Differentiation

Mesenchymal stem cells (MSCs) have generated a great deal of excitement and promise as a
Potential source of cells for cell-based therapeutic strategies, primarily owing to their intrinsic
ability to self renew and differentiate into functional cell types that constitute the tissue in which
they exist. MSCs are considered a readily accepted source of stem cells because such cells have
already demonstrated efficacy in multiple types of cellular therapeutic strategies, including
applications in treating children with osteogenesis imperfect [1], hematopoietic recovery [2], and
bone tissue regeneration strategies [3]. More importantly, these cells may be directly obtained
from individual patients, thereby eliminating the complications associated with immune rejection
of allogenic tissue. Despite diverse and growing information concerning MSCs and their use in
cell-based strategies, the mechanisms that govern MSC self-renewal and multilineage
differentiation are not well understood and remain an active area of investigation. Therefore,
research efforts focused on identifying factors that regulate and control MSC cell fate decisions
are crucial to promote a greater understanding of the molecular, biological and physiological
characteristic of this potentially highly useful stem cell type.

The multilineage differentiation potential of MSC populations derived from a variety of different
species has been extensively studied in vitro since their first discovery in 1960s [4]. These
studies demonstrate that populations of bone marrow derived MSCs from human, canine, rabbit,
rat, and mouse have the capacity to develop into terminally differentiated mesenchymal
phenotypes both in vitro and in vivo, including bone, cartilage, tendon, muscle, adipose tissue,
and hematopoietic-supporting stroma. The ability of MSCs to differentiate into a variety of
connective tissue cell types has rendered them an ideal candidate cell source for clinical tissue
regeneration strategies, including the augmentation and local repair and regeneration of bone,
cartilage and tendon.

Individual colonies derived from single MSC precursors have also been reported to be
heterogeneous in terms of their multilineage differentiation potential. For instance, Pittenger et
al. [5] reported that only one-third of the initial adherent bone marrow-derived MSC clones are
pluripotent (osteo/chondro/adipo). Furthermore, non immortalized cell clones examined by
Muraglia et al. [6] demonstrated that 30% of the in vitro derived MSC clones exhibited a tri-
lineage (osteo/chondro/adipo) differentiation potential, while the remainder displayed a bi-
lineage (osteo/chondro) or uni-lineage potential (osteo). These observations are consistent with
other in vitro studies using conditionally immortalized clones.
Several strategies have been employed to enhance and maintain the multilineage potential of
MSCs, such as culturing cells with specific growth factors, enriching cells prior to initial plating,
and/or culturing cells in a non-contact suspension culture configuration. However, the general
approach to the culture of MSCs involves isolating the mononucleated cells containing MSCs
from bone marrow aspirates and seeding these cells on tissue culture plates at a standard plating
density in a minimal essential medium base containing fetal bovine serum (FBS). Within 24-48
hours, nonadherent hematopoietic cells are removed, and the adherent cells are cultured and
passaged to expand the MSC population. Under this condition, cells can be expanded typically to
40 PDs until their growth rate is significantly reduced. Furthermore, addition of specific growth
factors in the MSC cultures has resulted in selective enrichment of different subsets of MSCs.

The inverse relationship: Adipogenesis and osteogenesis

The conventional view of linear hierarchical progression of stem cells from one differentiation
stage to the next during their phenotypic determination has been challenged by the recent
findings that adult stem cells can give rise to cells other than their residing tissues upon in vivo
transplantation [8-10]. Using an in vitro differentiation strategy, we recently showed that MSC-
derived, fully differentiated osteoblasts, adipocytes, and chondrocytes can switch their
phenotypes to other mesenchymal lineages in response to specific extracellular stimuli [11].
During the transdifferentiation process, extensive cell proliferation is observed and committed
cells lose their lineage-specific phenotype before resuming a cell state similar to primitive stem
cells, both in morphology and function. Furthermore, upon induction, these dedifferentiated cells
are able to acquire a new differentiated phenotype, that is, undergo redifferentiation. Taken
together, it is reasonable to conclude that both pre-committed progenitor cells and fully
differentiated cells retain the multipotentiality, and that their plasticity during ‘phenotypic
switching’ can be preserved during differentiation and be reaquired under defined, appropriate
microenvironmental circumstances, such as tissue repair and regeneration.
Osteoblasts, adipocytes and chondrocytes are believed to share a common mesenchymal
progenitor and these cells show the differentiation plasticity, resulting in the possible reciprocal
relationship of these lineages [12, 13]. In particular, osteogenesis is dominant over adipogenesis
in the bone marrow derived MSCs of younger adults, while this balance is usually reversed in the
bone marrow of older adults including patients with osteoporosis [14,15]. Such fate decisions are
regulated in part by transcription factors [15]. For example, researchers have previously reported
that mesenchymal differentiation can be switched from the adipocyte to the osteoblast lineage
simply by suppressing PPARc transactivation in cells [16]. In addition to transcription factors,
microenvironment may also regulate cell fate and subsequent tissue formation [17]. By
combining parameters such as cell density and the organization of multi-cellular structures in
three dimensional (3D) spaces, cell fate is likely determined through the compliance of local
environment and force equilibration through cell-cell/cell-matrix contacts.
An inverse relationship has been demonstrated between osteogenesis and adipogenesis [19]. As
an MSC in bone marrow stroma undergoes adipogenic differentiation, adipogenesis-related
genes [e.g., adipocyte P2 (aP2) lipoprotein lipase (LPL), and peroxisome proliferator–activated
receptor (PPAR)-_2] undergo up-regulation, whereas osteogenesis-related genes (e.g., collagen
type I and osteocalcin) are down-regulate [20]—a reciprocal relationship also manifested in
osteoporosis. During osteogenesis of MSCs, both in vitro and in vivo, is a well orchestrated
sequence of events, involving multiple steps and expression of various regulatory factors. During
osteogenesis, multipotent MSCs undergo asymmetric division and generate osteoprecursors,
which then progress to form osteoprogenitors, preosteoblasts, functional osteoblasts, and
eventually osteocytes [12]. This progression from one differentiation stage to the next is
accompanied by the activation and subsequent inactivation of transcription factors, i.e.,
Cbfa1/Runx2, Msx2, Dlx5, Osx, and expression of bone-related marker genes, i.e., osteopontin,
collagen type I, alkaline phosphatase, bone sialoprotein, and osteocalcin [21, 22]. Disruption of
the timely sequential expression of these genes results in the delay of the cell’s progression to the
osteoblast phenotype and the subsequent failure to form functional osteoblasts.

The habitual consumption of significant quantities of EtOH is recognized as a major factor for
osteopenia and increased fracture risk in both men and women [23, 24, and 25]. EtOH alters
osteoblast proliferation and function in vivo and in vitro [26, 27, 28] and inhibits the formation
of early osteoblast progenitors in murine and human bone marrow cell cultures [29] In addition
to a decrease in bone mass, osteoporosis is accompanied by an increase in marrow fat. This is
also observed in almost all conditions that lead to osteoporosis, such as ovariectomy [30], limb
immobilization [31], aging [32, 33], and excessive treatment with glucocorticoids. More
importantly, because the relationship between adipogenesis and osteogenesis is reciprocal, it is
possible that inhibition of marrow adipogenesis with a concomitant increase in osteogenesis
could provide a therapeutic strategy to either prevent further increases in adipocyte formation or
direct existing adipocytes to become more osteoblastic, with a resulting increase in functional
bone cells, thus reducing osteopenia.
Recently Gong and Wezeman et. all [34] demonstrated the inhibitory effect of EtOH on
osteogenesis in cultures of human MSCs (hMSCs). On the basis of these observations, we
hypothesized that EtOH promotes adipogenesis of hMSCs at the expense of osteogenesis. To test
this hypothesis, EtOH-induced adipogenesis was evaluated after induction of hMSCs in adipo
media. We studied three cases in the differentiation process of hMSCs induced by ethanol. We
also examined the Cytoskeleton organization and growth in the process of lineage commitment.

The doubling time is the period of time required for a quantity to double in size or value. When
the relative growth rate (not absolute growth rate) is constant, the quantity undergoes exponential
growth and has a constant doubling time or period which can be calculated directly from growth
rate. This time can be derived by dividing the natural logarithm of 2 by the exponent of growth,
or approximated by dividing 70 by the percentage growth rate unit for the exponential growth
equation, and its converse for exponential decay is the half-life.

Given two measurement of growing quantity, q1 at time t1 and q2 at time t2 and assuming a
constant growth rate, we can calculate the doubling time as ---

Td= (t1-t2)*log (2)/log (q2/q1)

 Where Td= doubling time
t1= initial time
t2= final time
q2= final concentration
q1= initial concentration

Hemocytometer

The hemocytometer is a device originally designed for the counting of blood cells. It is the
simplest, most convenient and cheapest means of accurately determining the numbers of cells in
a sample is to use a Hemocytometer and a microscope. A Hemocytometer is a specialised slide
that has a counting chamber with a known volume of liquid. It consists of a thick glass
microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved
with a laser-etched grid of perpendicular lines. The device is carefully crafted so that the area
bounded by the lines is known, and the depth of the chamber is also known. It is therefore
possible to count the number of cells or particles in a specific volume of fluid, and thereby
calculate the concentration of cells in the fluid overall.

Concentration of cells in original mixture =

3. .

Material and methods

Materials
DMEM (Dulbecco’s Modified Eagles Media, low glucose level)
Dulbecco’s phosphate-buffered saline (D-PBS)
0.05% trypsin/0.53 M ethylene diaminetetraacetic acid (EDTA)
Antibiotics/ Insulin
0.1% TritonX-100, 10% Formalin, 4% Paraformaldehyde( dituted in PBS from 10% stock)
Phalloidin TRITC (1mg/ml in DMSO), Oil Red stain, Giemsa Stain, Haemotoxylin stain

Cell preparation and culture methods

Human MSCs were harvested from patients undergoing routine treatment in hospitals. Primary
cultures of bone marrow cells were established by a modification of previously described
methods [35].
1. Bone marrow cells were released from the bony fragments into DMEM medium after
RBC cell lysis.
2. hMSCs were seeded in fibrotectin coated 12 well plate at density about 20,000
cells/cm.sq at 37°C in a humidified atmosphere containing 95% air and 5% CO2.
Medium was changed regularly, every three day interval.
3. When hMSC cultures become nearly confluent, cells were subcultured by rinsing with
calcium- and magnesium-free Hanks’ balanced salt solution, adding 3 ml of 0.05%
trypsin/0.53MEDTA, and incubating for 5 min at 37°C. The action of trypsin was then
inhibited by the addition of 3 ml of DMEM medium containing 10% FBS.

Ethanol Induction
When treatment started (day 0), hMSCs at passage 2 were cultured in the following three
conditions: (1) Normal control medium, (2) Adipo medium, (3) Adipo medium to which 50 mM
EtOH was added. The recovered cells from each patient were cultured in all three of the
experimental and control groups.
The concentration of EtOH (50 mM) was chosen according to previous reports that the half-
maximal effective concentration of EtOH to inhibit osteoblast proliferation in vitro is
approximately 50 mM, a level observed in actively imbibing alcoholic subjects [36].
Doubling time protocol
1. The biosafety cabinet was sterilized by UV radiator for 20minutes.
2. Medium, buffers were brought to room temperature.
3. Hela cells were passaged. Then trypsin was added to the flask and is allowed to stay for
2-3 minutes.
4. Serum was added to stop the reaction of trypsinizatin.
5. Hela cels were seeded in 12 well plates at the density of 2000 cells/cm.sq.
6. After every 12 hours, cells were agin trypsinized and the centrifuged and total increase in
cell count is measured by hemocytometer.

Cytoskeleton staining
1. The confluent cells were washed with PBS twice.
2. The cells were fixed with 4% paraformaldehyde at RT for 20 minutes.
3. Paraformaldehyde was removed and washed with PBS (5*5 min)
4. hMSCs were incubated with 0.1% Triton X-100 in PBS at RT for 15 min and then
washed with PBS (1*5 min).
5. Cells were incubated with 5% FBS in PBS for 1 hour at RT, covered with aluminum foil.
6. FBS was removed and it was incubated with diluted phalloidin 1:1000 at 4 degree celcius
overnight in dark.
7. Next day it was washed with PBS (5*5 mim) in dark and observed in microscope.

Oil red O Staining

For adipocyte visualization oil red staining was done after 24 days of incubation in all the control
groups and the different cases.
1. The media was removed and the cells were washed with PBS.
2. 10% formalin was used to fix the hMSCs , this was done for 30 minutes at room
temperature.s
3. The fixing solution was removed and washed with 60% isopropanol in water and stained
with oil red freshly prepared for 10 minutes.
4. Then it was washed with distilled water four times.
 5. For nucleus staining giemsa and haemotoxylin staining was carried out for 2 minutes.

Statistical analysis
Data are presented as mean _ SD. The mean average was determined in all the three cases and
Students T-test was performed. When a significant difference was determined and p _ 0.05 was
considered significant. Thus observation was statistically proved.

Results

Doubling time of HeLa cells

Following the above mentioned protocol, these observations were measured:

Time One side of Cell count Other side of Cell count Average
period Hemocytometer Hemocytometer total cell
(hr) count
Avg no. of cells (cells/sq.cm) Avg no. of cells (cells/sq.cm) (cells/sq.cm)
0 0 2000 0 2000 2000

12 0 2000 0 2000 2000

24 5 3676 4 2941 3308

36 25 18382 21 15441 16911

48 28 20588 36 26470 23529

60 30 22058 44 32352 27205

72 31 22058 50 36764 29411

1.a

1.b

1.c
Growth Curve of Hela Cells
40000
35000
30000
25000
Cells/Sq.cm

20000
15000 avg cell count
10000
5000
0
-5000 0 12 24 36 48 60 72
Time periods(hr)

Fig1: Doubling time of HeLa cells. Fig 1.a shows the 12 well plate with hMSCs seeded and the
cell count were observed till 72 hours. Fig 1.b shows the grids of hemocytometer where the cells
were counted. Fig 1.c shows the growth curve of the HeLa cells with std errors.

Calculations

Td= (t1-t2)*log (2)/log (q2/q1)

T= (36-24)*{log 2/log (16911/3308)}*3.4

= 21.6 hr

HeLa cells’ doubling time was observed and found to be 21.6 hours, the time in which the Hela
cells doubles its number and shows exponential pattern of growth.
Adipogenesis
During the differentiation process of hMSCs adipocytes in the medium containing 50 mM EtOH
was added showed more number of adipocytes as compared to the normal and adipo medium.
The major difference was noticed that adipocytes in the midum containing 50 mM EtOH were at
more developing phase or juveline stage. Whereas the adipocytes in the adipo medium were fully
developed after the 24 days of induction.

10X.A B

25X.A B

Fig 2
 40X.A B

Fig 2: Oil Red staining of hMSCs after 24- days. HMSCs were cultred in the normal medium (
control) 10x.A,B shows the 10X magnification;25X.A,B shows the hMSCs in 25X
magnification; 40X.A,B shows the 40X magnification.

10X.A B

25X.A B

Fig 3
40X.A B

Fig 3: Oil Red staining of hMSCs after 24- days. HMSCs were cultred in the adipo medium (
control) 10x.A,B shows the 10X magnification;25X.A,B shows the hMSCs in 25X
magnification; 40X.A,B shows the 40X magnification

10X.A B

25X.A B
 40X.A B

Fig 4: Oil Red staining of hMSCs after 24- days. HMSCs were cultred in the adipo medium after
treatement with50 mM EtOH. 10x.A,B shows the 10X magnification;25X.A,B shows the hMSCs
in 25X magnification; 40X.A,B shows the 40X magnification

Total cell (hMSCs) count*

Condition Normal media Adipo media Adipo media + ethanol

1 37248 34872 30757

2 42670 40937 41154

3 54150 46135 56965

4 56632 44362 45919

Average total cells 47675 41576.5 43698.75

*Human eye counting error

Number of Adipocytes in the eight fields

Condition Normal media Adipo media Adipo media+ ethanol

1
1 24 46
2
3 61 70
3
0 69 60
4
2 38 64
5
0 40 36
6
2 27 20
7
0 40 25
8
0 33 39

Total 8 332 360

Std. Deviation 1.195 15.775 18.338

Total Adipocytes
400

350

300

250

200 Total adipocytes

150
Graph1: Effect of EtOH on
100 MSCs in the adipogesis.
hMSCs cultutured in three
50
different cases.
0

Normal Adipo Adipo+EtOH

Condition Normal media Adipo media Adipo + ethanol media

Total cells 47675 41576.5 43698.75

Total Adipocytes
8 332 360

% Adipocyte conversion 0.016 0.77 0.82

Adipocyte conversion Percentage

1
0.9
0.8
0.7
0.6
0.5 % adipocyte conversion
0.4 Graph 2: Effect of EtOH on the
0.3 adipocyte conversion percentage
0.2 in three different conditions.
0.1
0

Normal Adipo Adipo+EtOH

T-test
Condition p-Value

Normal Vs Adipo media 4.28503E-06

Adipo Vs Adipo +Ethanol media 0.688552982

Normal Vs Adipo +Ethanol media 8.99263E-06

Oil red staining confirmed the adipogenic effect of EtOH. Human MSC cultures were stained
after treatment with 50 mM EtOH for 24 days. No Nile red– positive adipocytes were seen in
control– treated cultures at the time points (control versus EtOH; Figs. 2–4), indicating that
hMSCs do not undergo spontaneous adipogenic differentiation in the absence of induction. Small
lipid-containing Nile red–positive cells appeared after 10 days in Adipo and Adipo+EtOH. The
lipid droplets appeared in the periphery of the adipocytes in the induced cultures and began to fill
the cytoplasmic compartment in Adipo+ EtOH cultures (Adipo versus Adipo+ EtOH; Figs.3-4).
Although the percentage of adipocytes in Adipo cultures also increased with time, the change
was not as dramatic as that seen in the Adipo+ EtOH cultures. Further the T-test confirmed
statistically the NULL hypothesis that EtOH induces Adipogeneis, (0.688552982) P>0.05; Adipo
versus Adipo+EtOH cultutres.

Spectrometric analysis
Oil Red O stained lipid molecules (Adipocytes) in both cases of the culture were dissolved in
100% isopropanol. Absorbance was taken at 500nm using 96 well multiple plate reader
(TECAN)

Absorbance at 500nm

Adipo medium
O.D 0.061 0.092 0.0897 0.0903

O.D 0.0598 0.1123 0.0998 0.0868 Total Average

Average 0.0604 0.10215 0.09475 0.08855 0.086463

Adipo medium+ EtOH
O.D 0.0557 0.107 0.1014 0.0953

O.D 0.0564 0.1077 0.1099 0.0935 Total average

Average 0.05605 0.10735 0.10565 0.0944 0.090863

O.D
0.092
0.091
0.09
0.089 Graph3: shows the
0.088 comparative analysis of the
O.D
0.087 Absorbance.
0.086
0.085
1) Adipo medium
0.084
2) Adipo+EtOH
1 2
Cytoskeleton Staining

20X 50X

Fig 4: The cytoskeleton stained (Phalloidin TRITC) hMSCs cultured in the normal media. Two
different magnifications were viewed at 20X, 50X.

Conclusion
EtOH’s suppressive effects on osteogenesis have been documented in numerous experimental
animal studies; these effects seem to mimic the effects of EtOH on human skeletal metabolism.
EtOH-induced osteopenia reflects the inverse relationship between osteogenesis and
adipogenesis. Osteoporotic bone characteristically has an increased amount of marrow fat. In a
previous in vitro study [34], EtOH inhibited hMSC osteogenic differentiation by reducing
collagen type I synthesis and alkaline phosphatase activity. In addition, collagen type I gene
expression was down-regulated by EtOH. Because of the reciprocal relationship between
osteogenesis and adipogenesis, we hypothesized that EtOH might promote hMSCs
differentiation toward adipogenesis.
In this study, we examined the EtOH induced adipogenesis in the cultured hMSCs. EtOH might
induce spontaneous adipogenesis by shifting the lineage commitment into the more adipogenic
differentiation. Adipogenesis was morphologically evaualted by Oil Red staining. Adipocytes in
the culture treated with 50 mM EtOH outnumbered of adipocytes in the hMSCs culture with
Adipo medium. The adipocytes in the developing phase in the culture treated with 50 mM EtOH
supported the hypothesis that EtOH induces the adipogenesis after 24 days. These data, together
with our previous study on the inhibitory effect of EtOH on hMSC osteogenesis, indicate that
EtOH plays an important role in the balance between adipogenesis and osteogenesis, and this
might help to explain EtOH-related osteoporosis accompanied by increased marrow fat
accumulation.