Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

Abstract

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.

Sexual dimorphisms in adult human neural, mesoderm‐derived, and neural crest‐derived stem cells

“…We previously reported the presence of a particularly interesting population of NCSCs within the respiratory epithelium of the inferior turbinate in the human nasal cavity [78]. Namely, ITSCs gave efficiently rise to glutamatergic [41] and dopaminergic neurons [82] but also to mesodermal derivates like chondrocytes, adipocytes, or osteocytes [80,81,85,86]. Namely, ITSCs gave efficiently rise to glutamatergic [41] and dopaminergic neurons [82] but also to mesodermal derivates like chondrocytes, adipocytes, or osteocytes [80,81,85,86].…”

“…We demonstrated in several studies that such ITSCs are able to differentiate into a wide range of mesodermal and ectodermal cell types [78,81,84]. Namely, ITSCs gave efficiently rise to glutamatergic [41] and dopaminergic neurons [82] but also to mesodermal derivates like chondrocytes, adipocytes, or osteocytes [80,81,85,86]. Due to their extraordinary broad differentiation potential, NCSCs and particularly ITSCs seem an ideal model system for understanding potential sex-specific differences in endogenous differentiation and regeneration processes.…”

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells

“…These data further corroborate the CRM experiments (Figure ). Even though the special properties of ATI cells, that is, their exceedingly flat morphology, make it difficult to provide further distinguishing feature past day 3, several recent papers have demonstrated that CARS microscopy is well suited for following the differentiation of stem cells . To observe the gradual metamorphosis of ATII into ATI‐like cells on a larger scale, we finally performed live cell video imaging.…”

Label‐free quantitative chemical imaging and classification analysis of adipogenesis using mouse embryonic stem cells

“…To date, only a few studies of stem cell differentiation using CRS micro‐spectroscopy have been reported . In Ref.…”

“…. Hofemeier and co‐workers used Raman and CARS micro‐spectroscopy to identify early signs of calcium deposition during osteogenic differentiation of human stem cells . A hyperspectral CARS investigation of adipogenic and osteogenic differentiation of human mesenchymal stromal cells (MSCs) in Ref.…”