Detection of Osteogenic Differentiation by Differential Mineralized Matrix Production in Mesenchymal Stromal Cells by Raman Spectroscopy

<div><p>Mesenchymal stromal cells (MSCs) hold great potential in skeletal tissue engineering and regenerative medicine. However, conventional methods that are used in molecular biology to evaluate osteogenic differentiation of MSCs require a relatively large amount of cells. Cell lysis and cell fixation are also required and all these steps are time-consuming. Therefore, it is imperative to develop a facile technique which can provide real-time information with high sensitivity and selectivity to detect the osteogenic maturation of MSCs. In this study, we use Raman spectroscopy as a biosensor to monitor the production of mineralized matrices during osteogenic induction of MSCs. In summary, Raman spectroscopy is an excellent biosensor to detect the extent of maturation level during MSCs-osteoblast differentiation with a non-disruptive, real-time and label free manner. We expect that this study will promote further investigation of stem cell research and clinical applications.</p></div

Molecular biology detection of MSC-osteoblast differentiation.

<p>(A) Real-time PCR of OB-cadherin expression. (B) Western blot of OB-cadherin expression during osteogenic induction of MSCs. SaOS2 served as positive control. (C) The OB-cadherin expression level intensity analyzed by semi-quantitative method and was normalized to the intensity of SaOS2. (D) Alkaline phosphatase (AP) staining and von kossa (VK) staining.</p

Figure 3

<p>(A) Raman spectra of MSCs during osteogenic differentiation from 900 to 1800 cm⁻¹. (B) Magnified detail region for species from 900 to 1020 cm⁻¹ in stack diagram. OCP at 957 cm⁻¹ decreased upon osteogenic differentiation; β-TCP at 970 cm⁻¹ transiently appeared at Day 9 and HAP at 960 cm⁻¹ significantly increased after day9.</p

Figure 2

<p>(A) Background signal of Raman spectra. Magnified details of the region for control and cell species from 900 to 1020 cm⁻¹ in stack diagram.</p

Figure 5

<p>(A) Micrographs of MSCs treated with osteogenic media for 0, 3, 9, 15 and 24 days. (Scale bar: 100 µm) (B) Gene expression profiles of RUNX2, periostin, and type I collagen were detected in MSCs after Raman measurements by qPCR and normalized by internal and undifferentiated controls. Data are shown as mean ± SE (n = 3). (C) Alkaline phosphatase and Von Kossa staining of MSC-osteoblast differentiation. (D) Gene expression profiles of RUNX2, periostin, and type I collagen in staining samples were detected by qPCR and normalized by internal and undifferentiated controls. Data are shown as mean ± SE (n = 3).</p

Schematic diagram of the Raman platform set-up.

<p>Schematic diagram of the Raman platform set-up.</p

MiR-155 targeting SHIP1 under hyperstretch.

<p>Stretch-reduction of SHIP1 is reversed by anti-miR-155 (A), but not pre-miR-155 (C), compared with non-specific control anti-miR and control pre-miR, respectively. Relative intensity of SHIP1 protein expression was normalized to the expression ofβ-actin and static control. (C) Determination of SHIP1 is a direct target for miR-155 by using SHIP1 3′-UTR luciferase activity with/without anti-miR-155 treatment under hyperstretch. The changes in luciferase activity are relative to static control. Data represent mean ± s.e.m. *, P<0.05; n = 5.</p

Schematic representation of the role of hyperstretch and hMSC in pulmonary cell inflammation.

<p>Hyperstretch induces miR-155 to target SHIP1, which leads to the increases of JNK activation and consequential IL-8 secretion. Co-culture of hMSCs exhibits the anti-inflammation effects to reverse the hyperstretch-induced hBEC inflammatory responses.</p

Secretion of inflammation-related cytokines by hBECs under hyperstretch.

<p>(A) Cytokine levels in conditioned medium of hBEC under hyperstretch were measured using a human cytokine Multi-Analyst ELISA array and normalized to the conditioned medium of static control. (B) Hyperstretch regulation of pro-inflammatory IL-8 secretion in hBECs over 24 hour period of time. (C) Hyperstretch regulation of anti-inflammatory cytokine IL-10 secretion by hBECs and hMSCs. The changes in secretion level are relative to static control. Data represent mean ± s.e.m. *, P<0.05; n = 3.</p

The roles of IL-8 and IL-10 in hyperstretch-induced IL-8 secretion in hBEC/hMSC co-culture.

<p>(A) Schematic drawing of the top view of the stretching co-culture system. Measurement of IL-8 secretion levels in hBECs (first four bars) and in hBEC/hMSC (5^th and 6^th bar) after treatments with (B) exogenous IL-10, (C) IL-8 antibodies, (D) IL-8 receptor [IL-8R] blocking antibodies, (E) IL-10 antibodies, and (F) IL-10 receptor [IL-10R] blocking antibodies. The results demonstrated that hMSCs and IL-10 had similar effects, whereas IL-8 and hMSCs had contrary effects, on hyperstretch-induction of hBEC IL-8 secretion. The changes in secretion level are relative to static control. Data represent mean ± s.e.m. *, P<0.05; **, P<0.01 n = 5.</p