Author
Nicole J. Crane, Ph.D.

Naval Medical Research Center
Combat Casualty Care, 503 Robert Grant Avenue
Silver Spring, MD 20910

A Nondestructive Method for Monitoring in vitro Stem Cell Osteogenic Differentiation with Raman Spectroscopic Mapping

Introduction

Over the past few years, the discovery that stem cells can differentiate into various cell types has driven an increase in stem cell research. In turn, this has opened up new possibilities for regenerative medicine and gene therapies.

Bone marrow derived stem cells have the capacity to differentiate into osteoblasts, chondroblasts, and adipocytes. These cell types are responsible for production of bone, cartilage and adipose tissue respectively. Monitoring the differentiation of cells in vitro is challenging. Common methods include cell staining and sorting, but these techniques can be tedious and error-prone, as well as damaging to the cells.


Figure 1. Bone marrow derived stem cell differentiation into the osteoblastic lineage. Growth factors are secreted by cells to promote differentiation at various stages. Differentiation into a mature osteoblast is demonstrated by secretion of mineral crystals onto a protein matrix.¹

Here, Raman spectroscopic mapping provides a nondestructive method for monitoring in vitro stem cell osteogenic different-iation (the formation of osteoblasts – bone forming cells – Figure 1). Osteogenic differentiation is a stepwise process greatly influenced by soluble growth factors¹, and for this reason special osteogenic differentiation media are used to help mesenchymal stem cells (MSCs) differentiate into osteoblasts. First, the cells commit to the differentiation pathway and the first differentiation genes are induced. Next, the differentiation itself involves the induction of the entire differentiation gene set. Finally, the accumulation of gene-specific proteins marks cell maturation.² Figure 1 lists common gene expression profiles of the MSCs at various stages of osteogenic differentiation. Type I collagen expression begins as the cells differentiate from a preosteoblast into an osteoblast. Mature osteoblast differentiation, however, is denoted by mineral secretion onto a protein scaffold. Raman spectroscopy has the advantage of being able to detect not only changes in protein content and structure but also the deposition of mineral onto the protein matrix.


Figure 2. (left) Cell pellet in the bottom of the capillary. (middle) Raman image of cells in the capillary. (right) Raman spectrum representative of cells.

Raman monitoring of cell differentiation

To monitor the differentiation of the stem cells, approximately 5 × 10⁵ human mesenchymal stem cells were pelleted at the bottom of a 2.5 mm diameter quartz capillary (Figure 2, left) and cultured in the capillary for 21 days. Cells were fed with osteogenic differentiation growth media containing dexamethasone and ascorbic acid and the capillaries were incubated at 37°C and 5% CO₂, in 15 mL conical vials. One hundred microliters of growth media was replaced in the capillaries every 3-4 days. Each capillary was examined at six time points: day 0, day 3 (36 hours after addition of osteogenic media), day 7, day 10, day 15, and day 21. Each Raman map contained 12-15 mapping points (Figure 2, center), depending on the shape of the cell pellet in the bottom of the capillary.

Studies were conducted on a PerkinElmer^® RamanStation™ 400F with motorized stage using the following map- ping parameters: Wavelength range of 1800-500 cm^-1, 2 accumulations of 90 s acquisitions at each point in the map, 0.2 mm steps. Analysis time was kept short so as to minimize time samples were out of the incubator. Typical Raman bands observed in the spectra of the cells were 1004 cm^-1 (C-C ring breathing: phenylalanine/protein), 1305 cm^-1 (CH₂ deformation: protein), 1445 cm^-1 (CH₂ scissoring: protein), and 1660 cm^-1 (amide I: protein) in Figure 2 (right).³

When mineral deposition begins to occur, a Raman band at 960 cm^-1 (ν₁ P-O stretch: apatite-like mineral) can be observed. Once the mineral begins to mature, a Raman band at 1070 cm^-1 can also be observed (ν₃ PO₄^3- and ν₁ CO₃^2- stretch: apatite-like mineral). The mean spectra extracted from the Raman images at each time point are offset and overlaid in Figure 3, spectra are normalized using the 1550-1750 peak.


Figure 3. Mean Raman spectra of each observed time point during hMSC osteogenic differentiation. The black line indicates the 960 cm^-1 mineral band and the gray line indicates the 1070 cm^-1 mineral band.

The gradual appearance of the ν₁ P-O stretch at 960 cm^-1 band begins at day 7 and increases in intensity through day 21. Using more conventional techniques for cell culturing (well plates), mineralization is generally ob-served after two weeks of culturing. Here, mineralization is detected as early as day 7. Mineralization can then be directly monitored by calculating the mineral-to-matrix ratio (MTMR) for each spectrum. The MTMRs of each spectrum were calculated by dividing the 960 cm^-1 ν₁ P-O stretch vibrational band by the 1450 cm^-1 CH₂ scissoring vibrational band (Figure 4). It is clear that the MTMR, indicative of total mineral content, increases over time, as expected. The increased intensity of the 1070 cm^-1 band is indicative of carbonate ions being incorporated into the mineral crystal lattice. Carbonation of mineral tends to increase in normal bone as the mineral matures.⁴


Figure 4. Mineralization is detected as early as day 7 and is then directly monitored by calculating mineral to matrix ratio for each spectrum.

Conclusion

Determining the extent of osteoblast mineralization is generally carried out using staining techniques such as Alizarin Red staining. Staining, however, destroys the cells, takes more than an hour to process for each time point and requires the preparation of complete match-ing sets of samples for parallel RNA studies. Thus to monitor osteogenic differentiation and mineralization at different time points requires multiple cell cultures, and does not enable the monitoring of the same cells over time. Here, we present Raman spectroscopic mapping as a technique to non-invasively monitor mesenchymal stem cell differentiation into an osteoblastic lineage in less than an hour.

Raman offers more rapid sample analysis without burden- some staining, and halves the number of cell lines that must be prepared and maintained. Furthermore, with this method, the RNA and mineralization data are correlated, significantly improving the robustness of the study.

Future studies might show whether a single Raman analysis could simultaneously provide information on osteoblast mineralization and cell lineage.

References