Development of sphingosine kinase (SPHK) inhibitors and the role of sphingolipids in adult stem cell proliferation and differentiation 3

3.1.1.4 Stem Cells Osteogenic and Adipogenic Induction Culture Condition Culture media Supplements Normal growth DMEM+10% FBS +1% Penicillin and Streptomycin 10μM insulin, 200μM indomet

CHAPTER 3 ROLES OF SPHK INHIBITORS IN HUMAN BM-

AND AD- MSCS DIFFERENTIATION

Human stem cells are more and more becoming the research focus for their use in developmental biology and for their potential in clinical applications, to replace or replenish destroyed or dysfunctional tissue/organ However, one of the major risks using stem cells for therapy is their spontaneous differentiation ability to form teratoma

in the body (Wakitani et al., 2003; Vogel, 2005; Nussbaum et al., 2007; Hentze et al.,

2007) Therefore, promoting stem cells differentiation in a controlled manner will be highly desirable to reduce the risk of teratoma formation

Currently, there are several strategies to induce stem cells to differentiate along certain

lineages, introduced in Section 1.4.5.2 Exogenous Cytokines and Growth Factors,

Nonproteinaceous Cocktails, Scaffold, Co-culture, or Genetic Modification for MSCs Differentiation However, these methods share several common limitations such as

prolonged culture duration, high cost, inability to consistently differentiate stem cells along specific lineages, risk of pathogen transmission and the use of controversial genetic engineering techniques such as viral vector constructs to introduce proliferative and/or lineage-specific genes Therefore, it is of wide interest to obtain more knowledge

on the molecule(s) that promote stem cells differentiation in order to develop novel and safer strategies for stem cells differentiation

In our study, inhibition of the enzyme SPHK, which is tightly related to the cells proliferation, was evaluated as a new strategy to promote human MSCs differentiation

It is well known that proliferation and differentiation are two of the most fundamental

them that determines cell fate When the balance is broken, the cells would grow without differentiation, or commit into differentiation Based on the proliferative role of SPHK, which has been introduced in Chapter 1, we hypothesized that when suppressing SPHK activity in the stem cells, the balance between proliferation and differentiation could be lost, the cell growth was arrested, and the cells would then enter into certain

differentiation pathway(s) This hypothesis was supported by a recent study by Pébay et

al (2005), which showed that S1P could work synergetically with PDGF to promote

human embryonic stem cells proliferation, while the inhibition of SPHK by DMS, abolished the stemness maintained by S1P+PDGF and caused the cells to spontaneously differentiate, to unknown and uncharacterized lineage(s) However, no further research was carried out For example, it is not known what kinds of cells those human embryonic stem cells were differentiated into, or whether SPHK inhibition may aid the stem cells to differentiate into a specific cell-type(s)

In Chapter 2 I introduced the development of novel SPHK inhibitors, and the evaluation showed that the compounds generated were specific for SPHK1 Among these synthetic compounds, CP6 showed to be the best inhibitor candidate, in terms of the ability to penetrate into the cells and inhibit the endogenous SPHK1 and the good yield obtained from the chemical synthesis procedure Therefore, in this chapter, CP6 was selected to study the function of SPHK in human BM-MSCs and AD-MSCs differentiation, and the non-specific SPHK inhibitor, DMS, was used as a control Two differentiation pathways were chosen to study, namely, osteogenic and adipogenic differentiation, as these are well characterized pathways in human BM- and AD- MSCs differentiation

3.1 MATERIALS AND METHODS

All chemicals, if not specially mentioned, were purchased from Sigma-Aldrich, Singapore

3.1.1 Cell culture

3.1.1.1 Human AD-MSCs

First, human adipose tissue was obtained from Prof Dietmar W Hutmacher’s laboratory at the National University of Singapore, who had a protocol approved by the Institutional Review Board, National University Hospital, Singapore, for the procurement of human adipose tissue The adipose tissue was obtained from donors, after full written consent was obtained, who underwent elective liposuction Then, a

modified cell isolation protocol, based on Zuk et al.’s (2001) method, was used to

process the adipose tissues Briefly, the tissues were first washed three times with phosphate buffered saline solution (PBS) to remove the blood within the adipose tissues Tissues were then digested with 0.075% collagenase Type I (GIBCO #17101-015) for 2 hours at 37°C with occasional shaking The mixture was then spun down at 300g for 10 minutes The digested tissue was separated into three layers with the cells at the bottom layer The top layers were discarded and the cells were then re-suspended in culture media containing high glucose DMEM (GIBCO, #10569) supplemented with 10% heat-inactivated FBS (GIBCO), 1% of 2mM L-glutamine, 10mg/ml streptomycin and 10U/ml penicillin, and plated in 20 ml culture flasks (TPP, Trasadingen, Switzerland) Once the cells were attached to the culture flask, the medium was changed to remove the unattached cells Half of the growth medium was changed every three days Cells

3.1.1.2 Human BM-MSCs

Human BM-MSCs were a kind gift from Prof Michael Raghunath, who had purchased them from Cambrex (Cambrex, USA) Briefly, the cells were obtained from BM withdrawn from the posterior iliac crest of pelvic bone of normal volunteers (healthy males and non-pregnant females between the ages of 18 and 45 years old) Human BM-MSCs were cultured in low glucose DMEM (GIBCO, #10567), supplemented with 10% FBS, 1% of 2mM L-glutamine, 10mg/ml streptomycin and 10U/ml penicillin Cells were cultured in an incubator at 37°C, 5% CO2 in a water-saturated environment

3.1.1.3 Stem Cells Seeding Density

Unless otherwise stated, stem cell seeding density used was 5,000cells/cm2 in all of the experiments using human BM- and AD- MSCs

3.1.1.4 Stem Cells Osteogenic and Adipogenic Induction Culture

Condition Culture media Supplements

Normal

growth

DMEM+10% FBS +1% Penicillin and Streptomycin

10μM insulin, 200μM indomethacin, 1% Penicillin and Streptomycin Osteogenic

differentiation

DMEM+10% FBS +1% Penicillin and Streptomycin

0.01μM 1,25-dihydroxyvitamin D3, 50μM ascorbate-2-phosphate, 10mM ß-

glycerophosphate

3.1.1.5 Human Fetal Osteoblast Progenitor (hFOB) Cell Line

The hFOB cell line is a clonal, conditionally immortalized human fetal cell line that is capable of osteoblastic differentiation and bone formation It was used as a positive control in MSCs osteogenic differentiation investigations The cells were cultured in

RPMI 1640 supplemented with 10% FBS (GIBCO, Invitrogen Singapore), 2mM glutamine, 10U/ml penicillin and 10mg/ml streptomycin at 37C, 5% carbon dioxide in a humidified atmosphere

3.1.2 Cell Lysis

Cell lysates were prepared by lysing the cells in RIPA buffer (0.01M Tris-HCl [pH 7.4], 0.15M NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 1mM EDTA, 1mM phenylmethylsulfonyl fluoride) for one hour at 4°C

3.1.3 Staining

3.1.3.1 Alizarin Red S Staining

Alizarin Red S staining is widely used to evaluate the calcium-rich deposits generated

by cells In this study, the staining was used to detect the mineralization in human MSCs during osteogenic differentiation Briefly, the induced cells were fixed in 10% formalin overnight at 4°C Alizarin Red S working solution was prepared by dissolving

2 grams of Alizarin Red S powder (Sigma-Aldrich #05600) in 100ml of distilled water The pH of the working solution was adjusted to 4.1-4.3 using 0.5% ammonium After discarding the formalin and washed twice with distilled water, Alizarin Red S working solution was added to fully cover the fixed cells Cells were then incubated at room temperature for 5 minutes and checked under microscope for the orange-red color to develop Excess dye was removed and the cells were washed three times with distilled water Alizarin Red S staining in each sample was quantified by dissolving the staining

3.1.3.2 Oil Red O Staining

Oil red O staining is commonly used to identify the exogenous or endogenous lipid deposits In the present study, it was used to detect the adipogenic differentiation of the human stem cells Briefly, cells were fixed in 10% formalin overnight at 4°C Oil Red O stock solution was prepared by dissolving 0.7 gram of Oil Red O powder (Sigma-Aldrich #O-0625) in 200ml isopropanol, followed by filtration A working solution was prepared by mixing six parts of the Oil Red O stock solution and four parts of distilled water After the formalin was removed, samples were washed with 60% isopropanol, and Oil Red O working solution was added and incubated for 10 minutes at room temperature Following the incubation the dye was removed and the cells were washed three times with distilled water Oil Red O staining in each sample was quantified by dissolving the staining with 100% of isopropanol and detecting the absorbance at 500nm

3.1.3.3 Fluorescence Immunostaining

Fluorescence immunostaining was used to detect the expression of three typical osteogenic differentiation proteins: osteocalcin, osteopontin, and osteonectin In brief, cells cultured in different conditions were fixed in 100% methanol (in 48 well plates) at -20°C for at least 24 hours, blocked by 1% BSA in TBS-T (0.05%), and probed with primary and secondary antibodies. The primary antibodies against bone matrix proteins used were: rabbit anti-human osteocalcin (Chemicon, #AB1858) (1:500 dilution); rabbit anti-human osteopontin (Chemicon, #AB1870) (1:500 dilution), and rabbit anti-human osteonectin (Chemicon, #AB1858) (1:1000 dilution) The secondary antibody used was

goat anti-rabbit IgG (Pierce, #31460) (1:5000 dilution) Antibodies were all diluted in 1% BSA in TBS-T (0.05%)

3.1.4 Gene Level Expression Detection

3.1.4.1 RNA Extraction

RNA was extracted from different samples and purified using RNeasy Mini Kit (QIAGEN, #74106) according to the manufacturer’s instructions; purified RNA samples were stored at -80°C until further use

3.1.4.2 Reverse Transcriptional PCR

Purified RNA was reverse transcribed to cDNA using MuLV reverse transcriptase RNase H (Fermentas, #EP0451) according to the manufacturer’s instructions Based on the optimized conditions, initially, RNA was mixed with master mix 1 which contained Oligo dT primers and incubated at 75°C for 5 minutes, then immediately chilled on ice This is called the ‘first-strand reaction’ in RT-PCR The ratio of master mix 1 components used is shown below:

RNA 10ng Water (RNase free) (depends on RNA volume)

Afterwards, master mix 2 was prepared and added into each reaction tube Reactions were incubated at 37°C for 60 minutes, and heated at 95°C for 5 minutes to deactivate the reverse transcriptase This is the ‘second-strand reaction’ in the reverse transcription PCR The ratio of each component for master mix 2 is shown below

Component 1 reaction ( μl)

dNTP master mix (10Mm) 1

RNase free inhibitor 1 (1μl for 10 reactions)

5 x M-MuLV reaction buffer 4

M-MuLV reverse transcriptase 1 (1ul for 10 reactions)

Total 7ul

3.1.4.3 Real Time PCR

The quantitative real time PCR used in this study was followed the protocol developed

by Leong et al (2007) This is a novel method using linear double-stranded DNA

molecules as the standards instead of using the typical gene-in-plasmid format Standards for different genes were obtained from the serial dilutions of the cDNA got from the reverse transcription PCR The exact numbers of DNA copies in the standards was calculated by the method introduced in Section 3.1.4.3.2 below Three dilutions of the standards (such as 1x104copies/μl, 1x105copies/μl, and 1x106copies/μl) were used for testing the mRNA expression of each gene Thus, using the standards as controls, the copy numbers of mRNA expressed in the sample could be calculated More details are addressed below

3.1.4.3.1 Primers Design

Primers for genes of interest were designed according to their cDNA sequences from PubMed The uniqueness of the suitable pairs chosen was checked by “blast” (http://www.ncbi.nlm.nih.gov/blast/) All primers were purchased from Research BioLab (Singapore) With the designed primers, cDNA from different samples were amplified by PCR, and verified based on their size in a 2% agarose gel under UV illumination

The genes and corresponding primers information is listed below

Gene Primer sequence Product size Human

DNA molecules in the different samples were calculated based on the knowledge of the average mass of a base pair The calculation method is addressed below:

The average mass of a base pair in DNA is about 615 dalton according to their structures (see table below); a dalton (Da), named in honor of the British chemist John Dalton (1766-1844), is equivalent to 1/12 the mass of the 12C which equals 1.66053873E-27 kg, i.e., 1 Da = 1 atomic mass unit

Average Nucleotide Mass DNA Sub-Unit Da 10-21g Molecular Formula

dAdenosine Monophosphate (A) 312.2 0.5184 C10H11N5O5P

1) Since the PCR product is double strand DNA, and the designed size is 100bp, the

mass of one copy of DNA is: average mass/base pair x size of product = 615x100=61500 (Da), which is equivalent to 61500x1.66053873E-27≈1.02E-22 (kg) In other words, one molecule of a designed double strand DNA weighs around 1.02E-22

3.1.4.3.3 Quantitative Real Time PCR

A quantitative real time PCR using a thermocycler (Stratagene Mx 3000P) was performed on the samples using the primers designed, with serial dilutions of standards and a set of no-template-controls (NTC) to detect particular gene expression in different

samples, as previously described by Leong et al (2007) Briefly, QuantiTect SYBR

Green PCR kit was used for master mix of PCR reagents (QIAGEN, #204143) Thermal cycling conditions for PCR and dissociation routines were: 95°C for 10 minutes, 45

cycles of 94°C for 30 seconds, 60°C for 45 seconds, 72°C for 30 seconds, 95°C for 1 second, 60°C for 30 seconds, slow ramp up to 95°C at 0.5°C per second with continuous measurement, 95°C for 10 seconds, 25°C for 30 seconds, end (dissociation

phase is in italics) Results are presented by the DNA copy numbers in different genes

3.1.5 Quantification of the Stem Cell Growth (PicoGreen Assay)

Human BM- and AD- MSCs were quantified by detecting the total DNA in the samples

by using Quant-iT PicoGreen dsDNA Reagent (Molecular Probes, USA), which is an ultra-sensitive fluorescent nucleic acid stain for double-strand DNA, following the manufacturer instructions Briefly, cells were cultured in 48-well plates under different culture conditions When the cells are ready for testing their DNA components, cells were washed twice with 1x PBS, and 200μl of distilled water was added into each well and at least five rounds of freeze-thaw (-80°C and 37°C) were performed to lyse the cells thoroughly Cells were then centrifuged and the supernatants that contained DNA components were measured by PicoGreen

3.1.6 Statistical Analysis

Results are expressed as mean ± SD Significance between mean values was determined

by Student’s t-test Samples were analyzed by sample equal variance, and tailed distribution, with a value of P< 0.05 considered significant

SPHK expression difference in different cells

human ADSCs (donor1) 5.59719

human ADSCs (donor2) 6.877579

Figure 3.1 RNA expression of SPHK (1&2) in human BM- and AD- MSCs A,

RNA samples were extracted from the cells cultured in a normal growth media, and quantified using real time PCR SPHK1 and SPHK2 copies in 10ng of total RNA are

shown in; B, ratio of SPHK1/SPHK2 in different samples Results are the average + the

standard deviation of triplicate samples from at least three separate experiments

A

B

The results show that both human BM- and AD- MSCs express SPHK1 and SPHK2, however, SPHK1 shows a higher level of expression than SPHK2

3.2.2 Compounds Inhibition on Human MSCs Growth

3.2.2.1 Effects of Different Dosages of DMS and CP6 on Human BM- and AD- MSCs Growth

CP6 and DMS were first tested at different concentrations for their potential cytotoxic and/or anti-proliferative effects Six doses of DMS and CP6 (0.5μM, 1μM, 3μM, 5μM, 10μM, and 20μM) were used on human BM- and AD- MSCs and cell-growth was measured The results are shown in Figure 3.2

Effects of DMS/CP6 on human BM-MSCs growth

Figure 3.2 Effects of DMS/CP6 on human BM- and AD- MSCs growth Human

BM-MSCs (A) and AD-MSCs (B) were cultured in 10% FBS, supplemented with

different doses of DMS or CP6 Cells were cultured for four days and the cell number was measured by the PicoGreen assay Results are the average + the standard deviation

of triplicate samples from at least three separate experiments (*P<0.01 vs 0μM, i.e control w/o any DMS or CP6; **P<0.05 vs 0μM, i.e control w/o any DMS or CP6 Student’s t-test)

The results presented in Figure 3.2 show that human BM-MSCs are more vulnerable than AD-MSCs, when treated with DMS or CP6 More specifically, in human BM-MSCs (Figure 3.2A), DMS significantly inhibited stem cells growth at amounts of 3μM

or higher, and CP6 inhibited the stem cells growth at amounts of 10μM or higher However, for human AD-MSCs (Figure 3.2B), DMS showed to inhibit stem cells growth significantly only when at amounts of 5μM or higher, and CP6 inhibited stem cells growth at amounts of 20μM

To maintain similar experimental conditions for both human BM- and AD- MSCs, the following concentrations were chosen: 0.5μM and 1μM of DMS, 0.5μM, 1μM, and 5μM of CP6

Effects of DMS/CP6 on human AD-MSCs growth

In this test, DMS showed to inhibit cell growth at lower concentration than CP6 for both human BM- and AD- MSCs One of the possible reasons for this finding could be, that DMS has no selectivity for SPHK1 and SPHK2, as well as some other enzymes like DAGK and PKC (as shown in Chapter 2), whereas CP6 is very specific to SPHK1 So

in Figure 3.2, lower concentrations of DMS might inhibit the stem cells growth not only through inhibiting SPHK1, but possibly also SPHK2, and some other enzymes

3.2.2.2 Effects of DMSO on Human BM- and AD- MSCs Growth

Since both CP6 and DMS were dissolved in DMSO, the amount of DMSO used in the working solution of DMS and CP6 were also tested to detect whether it would affect human BM- and AD- MSCs growth The results are shown in Figure 3.3

A DMSO Function on human BM-MSCs growth

Figure 3.3 Effects of DMSO on human BM- and AD- MSCs growth Cells were cultured in 10% FBS, supplemented with different doses of DMSO Human BM-MSCs

(A) and human AD-MSCs (B) were cultured for four days and the cell number was

measured by PicoGreen assay Results are the average + the standard deviation of triplicate samples from at least three separate experiments (*P<0.01 vs DMSO ratio=0, Student’s t-test)

The maximum amount of DMSO ever used for dissolving DMS and CP6 in this research project was 0.04% (1:2500) The results presented in Figure 3.3 clearly demonstrate that the DMSO amount utilized in these experiments was completely safe,

as it did not inhibit at all cell growth compared with untreated cells (DMSO=0)

3.2.3 Function of CP6 and DMS in Human BM- and AD- MSCs Differentiation

Human BM- and AD- MSCs are multipotent cells that are capable of differentiating along osteogenic, chondrogenic, and adipogenic pathways, when stimulated under

appropriate conditions (Section 1.4.4 Clinical Potentials of MSCs) In this study, only

osteogenic and adipogenic differentiation pathways were studied due to the high cost

DMSO Function on human AD-MSCs growth

and limited numbers of the MSCs The culture conditions for these two differentiation

pathways were addressed in Section 3.1.1.4 Stem Cells Osteogenic and Adipogenic Induction Culture

In the following sections, human BM- and AD- MSCs osteogenic differentiation is addressed first, followed by their adipogenic differentiation study (Section 3.2.3.2

Function of the compounds in human BM- and AD- MSCs adipogenic differentiation)

3.2.3.1 Function of the CP6 and DMS in Human BM- and AD- MSCs Osteogenic Differentiation

In human BM- and AD- MSCs osteogenic differentiation study, hFOB cells were used

as control cells, as this cell line is capable of osteoblastic differentiation and bone

formation (Yen et al., 2007)

Human BM-MSCs, AD-MSCs, and the hFOB cells were cultured in 6-well plates (for detecting the RNA expression of several genes in osteogenic differentiation, by real time PCR), or in 48-well plates (for detecting the mineralization in different samples by Alizarin Red S staining) The cells growth media was changed to the differentiation

media (Section 3.1.1.4 Stem Cells Osteogenic and Adipogenic Induction Culture) on the

second day Culture media was changed every three days and the cells were cultured in the differentiation media for 14 or 28 days

3.2.3.1.1 Alizarin Red S Staining for Mineralization in Human MSCs Osteogenic Differentiation

Alizarin Red S stains the mineralization during stem cells differentiation, which is regarded as a late stage marker in the osteogenic differentiation pathway

In all our osteogenic differentiation samples, Alizarin Red S staining did not show a very positive red color under pH=4.1-4.3 However, when 200μl of 0.5% NH3OH was added into each well, the staining color became redder (as the pH became more alkaline), and more visible The staining was quantified by measuring the absorbance at 500nm, and the results are shown in Figure 3.4

Alizarin Red S Staining of DMS/CP6 treated hFOB cells in Osteogenic Differentiation

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

Alizarin Red S Staining of DMS/CP6 treated human BM-MSCs in Osteogenic

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

Alizarin Red S Staining of DMS/CP6 treated human AD-MSCs in

Osteogenic Differentiation (type 1)

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

Figure 3.4 Quantification of the Alizarin Red S staining Cells were cultured in normal growth media (ctrl), osteogenic differentiation media (OD), or osteogenic

differentiation media with different dosages of DMS or CP6 HFOB (A), human MSCs (B) and human AD-MSCs (C & D) were fixed and stained after 14 or 28 days of

BM-culture The staining was dissolved in 200μl of 0.5% NH3OH and the absorbance was

measured at 500nm Figure C shows the trend from at least two donors cells of human AD-MSCs Figure D demonstrates the trend from one donor cells of human AD-MSCs

Results are the average + the standard deviation of triplicate samples from at least three separate experiments (* P<0.01, ** P<0.05, Student’s t-test)

Figures 3.4A shows the staining from hFOB cells going through the osteogenic differentiation It is clear that the cells showed more staining when induced with the osteogenic differentiation media (OD), as compared with the non-induced sample (ctrl), and this finding is consistent in both day 14 and day 28 samples Interestingly, the cells induced with the osteogenic differentiation media supplemented with 0.5μM of DMS, had a higher level of staining (Figure 3.4A, OD+0.5μM DMS 28 days vs OD 28 days, P<0.01), indicating that 0.5μM DMS promoted more mineralization formation A similar trend was found in 0.5μM of CP6 treated osteogenic-induced cells (OD+0.5μM CP6) in both 14-day and 28-day samples However, in the cells induced with the

Alizarin Red S Staining of DMS/CP6 treated human AD-MSCs in

Osteogenic Differentiation (type 2)

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

osteogenic differentiation media together with 1μM of DMS, the staining in 28-day culture sample was lower than that from 0.5μM of DMS treated samples (Figure 3.4A, OD+0.5μM DMS 28 days vs OD+1μM DMS 28 days, P<0.05), and is similar to the osteogenic induced samples without compound treatment Similarly, 1μM of CP6 did not show to promote any enhancement on differentiation above the untreated samples

An interesting finding in the CP6 treated osteogenic-induced samples is that, after 14 days culture, 5μM of CP6 seemed to promote the osteogenic differentiation (OD+5μM CP6 14 days vs OD, P<0.01) Unexpectedly, in the sample cultured for 28 days, 5μM

of CP6 attenuated the osteogenic differentiation in osteogenic-induced cells cultured (OD+5μM CP6 28 days vs OD 28 days, P<0.01)

In osteogenic-induced hFOB cells, cultured for 14 days with 0.5μM DMS (OD+0.5μM DMS), 1μM DMS (OD+1μM DMS), or 1μM CP6 (OD+1μM CP6), the quantified staining showed to be similar to the osteogenic induced samples without DMS/CP6 (OD)

Figure 3.4B shows the osteogenic differentiation in human BM-MSCs under different conditions Similarly with what we got from hFOB test, human BM-MSCs induced with the osteogenic differentiation media showed more staining after 28 days culture compared to the non-induced control (OD 28 days vs ctrl 28 days, P<0.01) However, osteogenic-induced cells treated with 0.5μM of DMS, 0.5μM of CP6, or 1μM of CP6 for 14 days or 28 days, did not show much difference compared with the induced cells without inhibitors Interestingly and similarly with the hFOB test, 1μM of DMS and 5μM of CP6 inhibited the differentiation of the cells-induced for 28 days (OD+1μM

cultured with 0.5μM or 1μM of DMS or 0.5μM, 1μM or 5μM of CP6 for 14 days, did not show much difference when compared to the samples without inhibitors (OD 14 days)

When human AD-MSCs were studied for their osteogenic differentiation, cells from certain donors showed to respond positively (or non-negatively) to the osteogenic differentiation media, and these cells were categorized as type 1 AD-MSCs in this study Some cells from other donors showed to respond negatively to the osteogenic differentiation media, and these cells were categorized as type 2 AD-MSCs

Figure 3.4C shows the staining in type 1 human AD-MSCs osteogenic differentiation under different culture conditions The only conclusive finding in Figure 3.4C is that, the induced cells cultured with 0.5μM of DMS for 28 days showed more staining as compared with the induced sample without DMS/CP6 cultured for 28 days (OD+0.5μM DMS 28 days vs OD 28 days, P<0.05), which is consistent with what we got from the hFOB test

Type 2 human AD-MSCs osteogenic differentiation under different culture conditions is shown in Figure 3.4D In this type of AD-MSCs, the presence of 0.5μM of DMS increased the staining in both 14 days and 28 days culture as compared with the osteogenic-induced sample without inhibitors (OD+0.5μM DMS 14 days vs OD 14 days, P<0.05; OD+0.5μM DMS 28 days vs OD 28 days, P<0.01) Moreover, 0.5μM and 1μM of CP6 also increased the staining in the 28 days culture samples (OD+0.5μM CP6 28 days vs OD 28 days, P<0.05; OD+1μM CP6 28 days vs OD 28 days, P<0.05)

Similarly with the hFOB and human BM-MSCs results, 5μM of CP6 attenuated the staining in the induced cells cultured for 28 days (OD+5μM DMS 28 days vs OD 28 days, P<0.05) None of the induced cells cultured with 1μM of DMS, 0.5μM of CP6, 1μM of CP6, or 5μM of CP6 for 14 days showed any difference as compared with the induced sample without DMS/CP6 (OD 14 days)

3.2.3.1.2 Osteocalcin, Osteopontin, and Osteonectin Expression of DMS/CP6 treated Stem Cells in the Osteogenic Differentiation

Besides detecting the degree of the osteogenic differentiation of human BM- and AD- MSCs by Alizarin Red S staining, we also investigated the expression of differentiation-related genes in DMS/CP6-treated cells during the osteogenic differentiation

The expressions of osteocalcin, osteopontin, and osteonectin, generally regarded as

osteogenic differentiation markers (Leong et al., 2006b), were studied

RNA expression and protein level expression of these three proteins were investigated

by real time PCR and fluorescence immunostaining

3.2.3.1.2.1 RNA Expression of Osteocalcin, Osteopontin, and Osteonectin

Cells, cultured under the different culture conditions in our experimental set up, were collected after 14 or 28 days RNA samples and cDNA samples were prepared using the

methods described in Section 3.1.4 Gene Level Expression Detection Real-time PCR

was then performed to investigate each sample collected for the RNA expression of osteocalcin, osteopontin, and osteonectin in DMS/CP6 treated cells The results are summarized in Figure 3.5 (for osteocalcin), Figure 3.6 (for osteopontin), and Figure 3.7 (for osteonectin)

1) Osteocalcin Gene Expression

Osteocalcin gene expression of DMS/CP6 treated hFOB cells and human BM- and AD- MSCs in osteogenic differentiation is shown in Figure 3.5

Osteocalcin RNA expression of DMS/CP6 treated human BM-MSCs in

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

Figure 3.5 Osteocalcin RNA expression Cells were cultured in normal growth media (ctrl), osteogenic differentiation media (OD), or osteogenic differentiation media with different dosages of DMS or CP6 After 14 or 28 days culture, cells were lysed for total RNA extraction, and cDNA from each sample was used in real time PCR, for

detecting copy numbers of osteocalcin expressed in hFOB (A), human BM-MSCs (B) and human AD-MSCs (C & D) Figure C shows the trend from at least two donors cells

of human AD-MSCs Figure D demonstrates the trend from one donor cells of human

AD-MSCs Results are the average + the standard deviation of triplicate samples from

at least three separate experiments (* P<0.01, ** P<0.05, Student’s t-test)

Figure 3.5A shows the osteocalcin gene expression from hFOB cultured in the osteogenic differentiation-induction media and treated or not with DMS/CP6 The

Osteocalcin RNA expression of DMS/CP6 treated human AD-MSCs

(type 1) in Osteogenic Differentiation

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

OD+0.5uM CP6

OD+1uM CP6

OD+5uM CP6

days, P<0.01) The osteocalcin gene expression in the hFOB cells cultured in the osteogenic differentiation media supplemented with 0.5μM of DMS, showed more expression of osteocalcin in both 14 and 28 days samples, compared to the osteogenic-induced sample without DMS (OD+0.5μM DMS 14 days vs OD 14 days, P<0.01; OD+0.5μM DMS 28 days vs OD 28 days, P<0.01) Interestingly, cells cultured in the osteogenic differentiation media supplemented with 1μM of DMS expressed much less osteocalcin as compared with cells grown in the induction media but without DMS (OD+1μM DMS 14 days vs OD 14 days, P<0.01; OD+1μM DMS 28 days vs OD 28 days, P<0.01)

Cells cultured in the osteogenic differentiation media supplemented with 0.5μM of CP6 for 14 days showed high expression of osteocalcin (OD+0.5μM CP6 14 days vs OD 14 days, P<0.01) Whereas, cells cultured in the osteogenic differentiation media supplemented with 0.5μM of CP6 for 28 days did not show much difference in osteoclcin expression when compared to the samples without CP6 (OD+0.5μM CP6 28 days vs OD 28 days) Similarly with the effects of 1μM DMS on osteocalcin expression, 1μM and 5μM CP6 attenuated the osteocalcin expression significantly as compared with the induced control cells (OD+1μM CP6 14 days vs OD 14 days, P<0.01; OD+1μM CP6 28 days vs OD 28 days, P<0.01; OD+5μM CP6 14 days vs OD

14 days, P<0.01; OD+5μM CP6 28 days vs OD 28 days, P<0.01)

Figure 3.5B shows the osteocalcin expression of DMS/CP6 treated human BM-MSCs in the osteogenic differentiation Surprisingly, the osteocalcin in the cells cultured in the osteogenic differentiation media did not show an increased expression as compared

with the non-induced control (OD 14 days vs ctrl 14 days; OD 28 days vs ctrl 28 days) Moreover, in the cells cultured with the osteogenic differentiation media together with 0.5μM of DMS for 14 or 28 days, with 1μM of DMS for 14 or 28 days, with 0.5μM of CP6 for 14 or 28 days, or with 1μM of CP6 for 14 days, the osteocalcin expression did not show much difference as compared with the induced control at the same culture duration (OD+0.5μM DMS 14 days vs OD 14 days; OD+0.5μM DMS 28 days vs OD

28 days; OD+1μM DMS 14 days vs OD 14 days; OD+1μM DMS 28 days vs OD 28 days; OD+0.5μM CP6 14 days vs OD 14 days; OD+0.5μM CP6 28 days vs OD 28 days; OD+1μM CP6 14 days vs OD 14 days) However, in the cells cultured with the osteogenic differentiation media, together with 1μM of CP6 for 28 days, or with 5μM

of CP6 for 14 or 28 days, the osteocalcin expression was even lower than that in the induced cells without inhibitors (OD+1μM CP6 28 days vs OD 28 days, P<0.01; OD+5μM CP6 14 days vs OD 14 days, P<0.01; OD+5μM CP6 28 days vs OD 28 days, P<0.01)

Figure 3.5C shows the osteocalcin gene expression of DMS/CP6 treated type 1 human AD-MSCs in the osteogenic differentiation Cells cultured in the osteogenic differentiation media expressed more osteocalcin mRNA as compared with the non-induced control (OD 14 days vs ctrl 14 days, P<0.05; OD 28 days vs ctrl 28 days, P<0.05) Cells cultured in the osteogenic differentiation media together with 0.5μM of DMS showed more osteocalcin expression only after 28 days of culture, but not after 14 days, as compared with the induced sample without inhibitor (OD+0.5μM DMS 14

DMS 28 days vs OD 28 days, P<0.01) Osteogenic-induced cells grown in the presence

of 1μM of DMS also expressed more osteocalcin at 28 days of culture, but not after 14 days of culture, as compared to the induced samples without inhibitor (OD+1μM CP6

14 days vs OD 14 days, P≈0.16, not significant and not shown in the figure; OD+1μM CP6 28 days vs OD 28 days, P<0.01) Interestingly, 0.5μM of CP6 triggered more osteocalcin expression both after 14 days and 28 days of culture, as compared with the induced sample without inhibitor (OD+0.5μM CP6 14 days vs OD 14 days, P<0.05; OD+0.5μM CP6 28 days vs OD 28 days, P<0.01) However, in the presence of 1μM of CP6, cells cultured in the osteogenic differentiation media, showed more osteocalcin expression only at the 28 days culture (OD+1μM CP6 14 days vs OD 14 days; OD+1μM CP6 28 days vs OD 28 days, P<0.01) In contrast, cells cultured in the osteogenic differentiation media supplemented with 5μM of CP6, did not show much difference on the osteocalcin expression levels, compared with the induced samples without inhibitor (OD+5μM CP6 14 days vs OD 14 days; OD+5μM CP6 28 days vs

a decreased levels of osteocalcin expression after 14 days culture, as compared with the induced sample without DMS (OD+1μM DMS 14 days vs OD 14 days, P<0.05) However, after 28 days culture, the cells cultured in the osteogenic differentiation media containing 1μM of DMS expressed similar amount of osteocalcin the cells cultured in the osteogenic differentiation media without DMS (OD+1μM DMS 28 days vs OD 28 days) Interestingly, cells cultured in the osteogenic differentiation media supplemented with 0.5μM of CP6 showed to have an increased osteocalcin expression in both 14 and

28 days cultured cells (OD+0.5μM CP6 14 days vs OD 14 days, P<0.01; OD+0.5μM CP6 28 days vs OD 28 days, P<0.05); 1μM of CP6 did not show much effect on the osteocalcin expression during the cells osteogenic differentiation (OD+1μM CP6 14 days vs OD 14 days; OD+1μM CP6 28 days vs OD 28 days) However, 5μM of CP6 inhibited the osteocalcin expression in the 28 days induced cells (OD+5μM CP6 28 days vs OD 28 days, P<0.05), but not in the 14 days induced cells (OD+5μM CP6 14 days vs OD 14 days)

2) Osteopontin Gene Expression

Osteopontin gene expression of DMS/CP6 treated hFOB cells and human BM- and AD- MSCs in osteogenic differentiation is shown in Figure 3.6