Effects of donor characteristics and ex vivo expansion on canine mesenchymal stem cell properties: implications for MSC-based therapies

Abstract

Clinical trials utilizing bone marrow-derived mesenchymal stem cell (BM-MSC) therapies show promise for treating a variety of pathologic conditions. Paramount to optimization of such cell-based therapies is a thorough understanding of MSC biology. Despite the tremendous potential that exists for the clinical use of canine BM-MSCs in veterinary medicine, as well as in preclinical studies for human medicine, relatively little information exists regarding basic biological properties of the cells. In this study, we compared the importance of donor characteristics (age and harvest site) and ex vivo expansion on canine BM-MSC frequency (CFU-f) and differentiation potential. Advancing age was found to have a negative effect on CFU-f as well as osteogenic potential. Site of harvest was also found to have significant effects on MSC properties. MSCs obtained from the humerus were found at the lowest frequency and were least osteogenic compared to those harvested from the tibia, femur, and ilium. Osteogenic potential diminished significantly by the third passage. These results suggest important donor parameters and culture effects to consider in translational studies examining MSC-based regenerative medical strategies.

Figure 1

Multipotential differentiation of canine bone marrow-derived mesenchymal stem cells. Osteogenesis. (A) Bone marrow mesenchymal stem cells (BM-MSCs) maintained a typical fibroblastic phenotype in control medium and showed no Alizarin red S staining. In contrast, cells cultured under osteogenic conditions (Induced) formed nodules and deposited a calcium-rich extracellular matrix as evidenced by Alizarin red S staining. (B) In addition, osteogenic cultures showed induction of bone sialoprotein (BSP) and osteocalcin (OCN) relative to control cultures (day 21 postinduction). Adipogenesis. (C) Oil Red O staining revealed cytoplasmic lipid inclusions in cells cultured in the presence of adipogenic inducers (Induced), as compared to cells cultured under control conditions (day 21). (D) mRNA expression analysis for adipogenic markers at the corresponding time point shows induction of peroxisome proliferator-activated receptor γ (PPARγ), lipoprotein lipase (LPL), and fatty acid binding protein-4 (FABP4) in induced cultures relative to control cultures. Chondrogenesis. (E, F) Alcian blue staining reveals a proteoglycan-rich matrix is secreted by BM-MSCs cultured under chondrogenic conditions (day 21) compared to control growth medium (E) with a corresponding induction of the chondrogenic markers, Aggrecan, type II collagen (Col2) and sex-determining region Y box 9 (Sox9) (F).

Figure 2

Effect of donor age and site of harvest on CFU-f and proliferation of BM-MSCs. (A) MSC frequency per 2 × 10⁶ of bone marrow mononuclear cells (BMMCs) in bone marrow samples [as determined by Colony forming unit-fibroblastic assay (CFU-f)] was significantly reduced in samples harvested from aged compared to young dogs (*p ≤0.01). Data from all sites were pooled for analysis of the effect of age on CFU-f, such that n = 31 and 21 for young and aged dogs, respectively. Each box represents the interquartile range (25th to 75th percentiles). The horizontal line within each box represents the median. (B) In contrast, donor age had no effect on canine MSC proliferation (data from all sites were collected and pooled where n = 40 and 25 for young and aged dog samples). Data are presented as MTS activity in day 6 cultures of MSCs obtained from young and aged dogs (plated at identical initial plating density). (C) Representative images of MSC cultures stained with giemsa stain for CFU-f assays, showing differences in CFU-f frequency in BMMCs harvested from the femur, humerus, ilium, and tibia of a young dog. (D) Quantitative analysis of CFU-f by harvest site shows an increase in frequency of MSCs harvested from iliac samples compared to the other sites. Two-way ANOVA confirms that site of harvest has a significant impact on CFU-f numbers (p < 0.05). (E) Proliferation in MSC cultures obtained from the tibia is significantly increased relative to that in cultures harvested from either the femur or humerus (*p≤0.01). Data are presented as mean MTS activity in day 6 cultures of MSCs obtained from the femur, humerus, ilium, and tibia in young dogs (plated at identical initial plating density).

Figure 3

Osteogenesis is diminished in MSCs harvested from aged compared to young dogs. First passage cultures of MSCs obtained from bone marrow of young and aged donors were cultured in control and osteogenic media. The effect of age on MSC response to osteoinduction was assessed by: quantitating alkaline phosphatase (AP) activity, osteogenic marker induction and mineralization. (A) Following 6 days of osteogenic induction, AP activity was determined and normalized on the basis of cell number. Each box represents the interquartile range (25th to 75th percentiles). The horizontal line within each box represents the median. Comparison of AP activity normalized to cell number revealed a significant decrease of AP in aged dog samples cultured under osteogenic conditions relative to those obtained from young dogs (*p < 0.05). (B) MSCs harvested from the femoral BM cavity of young and aged donors cultured in osteogenic conditions showed mRNA induction of both bone sialoprotein (BSP) and osteocalcin (OCN) compared to control cultures (14 and 21 days after induction, respectively), although cultures of MSCs from aged donors had a diminished response to osteo inducers relative to those harvested from young donors. Fold induction of OCN expression is significantly diminished in MSCs harvested from aged dogs relative to young dogs (*p < 0.05). (C) Analysis of quantified Alizarin red S extracted from the matrix of femoral bone marrow MSC cultures from young and aged donors cultured under either control or osteogenic conditions for 14 days reveals a significant effect of age on matrix mineralization. MSCs from both young and aged dogs respond to osteoinducers but those from aged donors are significantly less responsive than those from young donors (*p < 0.05). (D) Alizarin red S staining of mineralized matrix in representative cultures from a young and an aged donor showing increasing deposition of a richly mineralized matrix in the young donor cells as compared to aged donor cells at 7, 14, and 21 days. Mineralization also increases over time in aged dog MSC cultures, albeit at a diminished amount relative to young dogs at corresponding time points.

Figure 4

Site of harvest influences osteogenic potential of canine MSCs. A comparison of osteogenic potential of MSCs harvested from the femur, humerus, ilium, and tibia of young dogs reveals that humeral-derived MSCs are least responsive to osteoinducers. (A) AP activity is significantly elevated in osteogenic-induced cultures of MSCs harvested from the tibia relative to those from the humerus at day 6 postinduction (*p ≤ 0.01). Corresponding controls are shown to confirm minimal to no AP activity in these cultures and therefore the absence of differentiated cells under noninducing conditions. (B) Effect of site on induction of BSP and OCN mRNA expression in response to osteoinducers was assessed in MSC cultures at day 14. Values are set relative to BSP and OCN expression in humerus-derived MSC cultures, highlighting consistent increases in osteogenic marker expression in other harvest sites relative to the humerus. (C) Alizarin red S staining showing typical induction of matrix mineralization in MSC cultures (day 7) from a representative young donor. An increase in intensity of Alizarin red S stain is seen in cultures of MSCs harvested from the ilium and tibia relative to the femur and humerus. (D) Site of harvest was found to have a significant effect on mineralization of MSC cultures (p < 0.05). Cultures of MSCs harvested from the ilium and tibia were found to have the greatest mineralization while those from the humerus had the least mineralized matrix deposited. Multiple comparison analysis reveals a significant elevation in extracted Alizarin red S in cultures of MSCs from the ilium relative to the humerus.

Figure 5

Osteogenic differentiation is diminished with increasing passage in canine MSC cultures. Increasing passage was found to have a significant negative effect on AP activity within and mineralization of canine MSC cultures (p ≤ 0.01 for both). (A) Analysis of AP activity in day 6 osteogenic cultures of MSCs harvested from the femoral bone marrow of both young and aged dogs reveals diminishing AP activity with increasing passage. AP activity in aged dog MSC cultures are diminished in comparison to corresponding cultures from young dogs at each passage. A significant decrease in AP activity occurs by the third passage in young dogs and by the second passage in aged dogs (*p < 0.05). (B) Quantitation of Alizarin red S staining in day 7 cultures of MSCs harvested from femoral bone marrow of young dogs reveals diminished capacity for mineralization with increasing passage number. Mineral deposition in Passage 1 (P1) cultures is significantly greater (*) than in all later passages examined (P1 vs. P2–P4; *p ≤ 0.01). (C) Images of MSC cultures (day 21) stained with Alizarin red S from an individual young donor reveals that mineralization occurs throughout P1 cultures of MSCs but is restricted to smaller nodules in higher passage cultures (from left to right, P1 > P2 > P3 ~ P4).