Osteogenic Differentiation

Osteogenic differentiation was determined by Alizarin Red S labelling and quantified by the extraction using 1% CPC solution and measurement of the absorbance of this dye. A standard curve (not shown) was obtained by serial dilution of known concentrations of Alizarin Red S (ARS) dye (Sigma-Aldrich, UK). It was linear throughout the range of dye concentrations used. Therefore, absorbance values were used as a direct measure of ARS concentration.

BDM1 medium (containing ascorbic acid) was toxic to cells, and therefore results from these were not taken into account. For cells treated with BDM2 bone differentiation medium, the formation of bone nodules is clearly apparent in the fhMSC line 40mm. No such nodules were seen in 24XY fhMSCs (not shown) or hNSC negative controls (Figure 3-9).

Both hWJ cell lines (25-10-04 and 27-10-04) responded to BDM2 treatment (Figure 3-10A & B), with maximal Alizarin Red labelling at 2-3 weeks of differentiation culture.

Amounts of stain extracted from untreated hWJ cells remained constant throughout. hWJ cells treated with BDM1 were much lower than untreated ones at all timepoints except at 1 week.

At this first timepoint, hWJ cells in BDM1 (containing ascorbic acid) were higher than either untreated ones or those in BDM2 (containing ascorbate-2-phosphate, a more stable form of ascorbic acid). The latter were also lower than untreated controls.

In order to determine the effects of culturing hWJ cells in mitogen-supplemented medium on their osteogenic differentiation potential, hWJ-18-2-05 cells were cultured in D10/SFEM (D10 medium + EGF + FGF2 + B27 without retinoic acid) before inducing bone differentiation in BDM1 and 2. Untreated cells (also cultured in D10/SFEM) were used as negative controls. The amount of ARS extracted from stained cells was compared to hWJ-18-2-05 cells grown in D10 medium and induced to differentiate in bone cells (Figure 3-10C).

Culturing in D10/SFEM compared to D10 medium reduced the amount of calcium-rich extracellular matrix deposited by the cells. Although BDM2-induced osteogenic differentiation (as determined by dye extraction) did take place in hWJ cells previously cultured in the mitogen-supplemented medium, this was significantly different from those grown in D10 (Figure 3-10C).

A line of fhMSCs (40mm) was used as a positive control for bone differentiation (Figure 3-10D). There was an increase over time in both untreated (control, DMEM/10% FBS) cells and those treated with BDM2. fhMSCs treated with BDM1 remained much lower than untreated ones at all timepoints, due to the toxicity of this medium and consequent cell death.

Whilst the amount of stain extracted from untreated cells fell after 3 weeks, it continued to rise for those in BDM2.

hNSCs were used as a negative control for bone differentiation (Figure 3-10E). Although there was a small increase in the amount of Alizarin Red dye extracted from cells treated with BDM2 and untreated cells (control, DMEM/10% FBS), this was much lower than the response of fhMSCs and hWJ cells treated in the same way – there was no statistically significant difference in the amount of dye extracted from treated and untreated hNSCs at week 3 (Figure 3-10E). The amount of Alizarin Red dye extracted from hNSCs treated with BDM2 were lower or equal to untreated hNSCs at all timepoints except at 4 weeks, when BDM2-treated hNSCs were more heavily stained than control (untreated, in D10 medium) hNSCs. The amount of Alizarin Red dye extracted from hNSCs treated with BDM1 remained much lower than untreated hNSCs at all timepoints.

Figure 3-9 : Phase-contrast photomicrographs of fhMSCs (40mm), hWJ cells (hWJ-25-10-04) and hNSCs exposed to osteogenic differentiation medium BDM2. Bone nodules (dark rim and paler centre) were only seen in BDM2 and only in fhMSCs. Bar is 100µm.

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hWJ-25-10-04 p#6 - Bone Differentiation

hWJ-27-10-04 p#6 - Bone Differentiation

1 2 3 4

hWJ-18-02-05 - Bone Differentiation

Culture Condition prior to Differentiation

ARS Absorbance @ 562nm

C

p<0.0001, S p<0.0001, S

p=0.0156, S

40mm fhMSCs - Bone Differentiation

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Figure 3-10 A-E : Alizarin Red S (ARS) stain extracted from cells treated for up to 4 weeks in bone differentiation medium (BDM2). Readings were obtained from triplicate wells which were further divided into triplicates for absorbance measurement at 562nm.

Osteogenic differentiation of hWJ-25-10-04 (A), hWJ-27-10-04 (B), hWJ-18-2-05 cells (C, 3 weeks only), 40mm fhMSCs (D) and hNSCs (E) in BDM1 and BDM2 for 4 weeks, as determined by Alizarin Red S (ARS) absorbance measurements after extraction from stained cells. A One-Way ANOVA was carried out followed by a Bonferroni Multiple Comparison Post-Test to compare untreated (control) cells at different timepoints. No significant difference between untreated hWJ-25-10-04 cells at different timepoints (p>0.05). There was a significant difference only between untreated hWJ-27-10-04 cells at 1 and 4 weeks (p<0.01), and 2 and 4 weeks (p<0.001). Except at 1 and 2 weeks, amounts of due extracted from untreated fhMSCs were significantly different from each other between timepoints (p<0.01). BDM2-treated and untreated cells were compared with untreated controls at 3 weeks using an unpaired two-tailed T-test. S, Significant; NS, Non-Significant. n=3.

R197VM hNSCs - Bone Differentiation

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Summary :

hWJ cells and fhMSCs have osteogenic differentiation potential.

3.4.2 Fat Differentiation

Adipogenic differentiation was determined by Oil Red O (ORO) labelling and quantified by the extraction using 100% isopropanol and measurement of the absorbance of this dye. A standard curve (not shown) was obtained by serial dilution of known concentrations of Oil Red O (ORO) dye (Sigma). It was linear throughout the range of dye concentrations extracted from stained cell samples (corresponding to absorbance values of up to 0.75 units). Therefore, absorbance values were used as a direct measure of ORO concentration.

Similarly to 40mm fhMSCs, intracellular lipid globules were only seen in hWJ cells treated with FDM only (Figure 3-11). In terms of ORO accumulation, hWJ-27-10-04 responded best to the FDM/FMM fat differentiation protocol (Figure 3-12B). Although there was a gradual increase in the amount of ORO extracted from hWJ-25-10-04 cells treated using the same protocol, the control levels were unusually high compared to treated conditions (Figure 3-12A).

Using the same measure of adipogenic differentiation, 40mm fhMSCs responded only to the FDM/FMM protocol (Figure 3-12D), although there were no typical adipogenic cell morphologies using this protocol. These intracellular fat globules were only seen in the FDM protocol (Figure 3-11), even though the amount of ORO stain extracted from cells treated with FDM only was lower than untreated fhMSCs at all timepoints (Figure 3-12D).

As measured by the amount of ORO extracted, hNSCs responded to both fat differentiation protocols, even though their morphological characteristics were not the same as fhMSCs treated in the same way (Figure 3-11). hNSCs treated with FDM with no alternation with FMM medium accumulated more total lipid at all timepoints than those that were alternated between the two media (Figure 3-12E). Untreated hNSCs accumulated some lipid over 4 weeks.

Figure 3-11 : Phase-contrast photomicrographs of fhMSCs (40mm), hWJ cells (hWJ-25-10-04) and hNSCs exposed to adipogenic differentiation media. Intracellular lipid globules were only seen in FDM. Bar is 100µm.

FDM