Structural investigation of donor age effect on human bone marrow mesenchymal stem cells: FTIR spectroscopy and imaging

Abstract

Stem cell studies hold enormous potential for development of new therapies for tissue regeneration and repair. Bone marrow mesenchymal stem cells (BM-MSCs) can differentiate into a variety of non-hematopoietic tissues and contribute maintenance of healthy hematopoiesis by providing supportive cellular microenvironment into BM. Here, we investigated age-related differences in BM-MSCs by using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and FTIR imaging together with hierarchical clustering as a novel methods to clarify global alterations in the structure and function of macromolecules in characterized BM-MSCs of different aged donors. The results may contribute to identification of age-related new molecular marker(s) to determine the effects of donor age on MSCs. The spectral results reflected that there were significant increases in the concentration of saturated lipids, proteins, glycogen, and nucleic acids in children and adolescent group BM-MSCs when compared to the infants and early and mid adults. The concentration of mentioned macromolecules in adult (early and mid) BM-MSCs were significantly lower than the concentrations in the children and adolescents. These results were attributed to the increase in the proliferation activity in younger BM-MSCs. The distribution of macromolecules into the cells was shown as in the form of chemical maps by FTIR imaging, and the results are in agreement with the ATR-FTIR spectroscopy results. The cellular activity degree was determined by the thiazolyl blue tetrazolium bromide (MTT) proliferation assay to support ATR-FTIR spectroscopy results. BM-MSCs of five different age groups were discriminated by making the hierarchical cluster analysis where the spectral data according to alterations in structure and composition of macromolecules were considered.

Fig. 1

The results of surface antigen profiles of healthy BM-MSCs belonging to five different age groups

Fig. 2

a Oil Red O staining of healthy P3 BM-MSCs at the end of 21 days—infant MSCs (a), children MSCs (b), adolescent MSCs (c), early adult MSCs (d), and mid adult MSCs (e) (magnification = ×20). b Alizarin Red staining of healthy P3 BM-MSCs at the end of 21 days—infant MSCs (a), children MSCs (b), adolescent MSCs (c), early adult MSCs (d), mid adult MSCs (e) (magnification = ×20)

Fig. 3

The representative infrared spectra of healthy BM-MSCs from different age donors. Green line represents infant BM-MSCs; blue line represents children BM-MSCs; pink line represents adolescent BM-MSCs; red line represents early adult BM-MSCs; and black line represents mid adult BM-MSCs in the 3,800–800 cm⁻¹ region (the spectra were normalized with respect to the amide A band) (color figure online)

Fig. 4

The representative infrared spectra of healthy BM-MSCs from five different age groups in the 3,000–2,800 cm⁻¹ region. Green line represents infant BM-MSCs; blue line represents children BM-MSCs; pink line represents adolescent BM-MSCs; red line represents early adult BM-MSCs; and black line represents mid adult BM-MSCs (the deconvolved spectra were normalized with respect to the amide A band) (color figure online)

Fig. 5

The representative infrared spectra of healthy BM-MSCs from five different age groups in the 1,800–800 cm⁻¹ region. Green line represents infant BM-MSCs; blue line represents children BM-MSCs; pink line represents adolescent BM-MSCs; red line represents early adult BM-MSCs; and black line represents mid adult BM-MSCs (the spectra were normalized with respect to the amide A band) (color figure online)

Fig. 6

Spectral image maps that reflect distribution of lipids, proteins, and nucleic acids in the BM-MSCs from five different age groups. These maps were derived respectively by taking the peak integrated areas of a CH₂ antisymmetric stretching bands of lipids, b amide I band of proteins, and c PO₂ ⁻ antisymmetric stretching bands of nucleic acids

Fig. 7

Hierarchical cluster analysis performed on the vector-normalized spectra of BM-MSCs of infants, children, adolescents, early adults, and mid adults and resulting from Ward’s algorithm. The study was conducted in the a 1,800–800 and b 3,000–800 cm⁻¹ spectral regions