MSC frequency correlates with blood vessel density in equine adipose tissue

Abstract

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to develop into different mature mesenchymal cell types. They were originally isolated from bone marrow, but MSC-like cells have also been isolated from other tissues. The common feature of all of these tissues is that they all house blood vessels. It is, thus, possible that MSCs are associated with perivascular locations. The objective of this work was to test the hypothesis that MSCs are associated with blood vessels by verifying if MSC frequency positively correlates with blood vessel density. To this end, samples from highly and poorly vascularized adipose tissue sites of two equine donors were collected and processed for histology and cell isolation. MSC frequency in these samples was estimated by means of CFU-F assays, which were performed under MSC conditions. Culture-adherent cells from equine adipose tissue and bone marrow were culture expanded, tested for differentiation into mesenchymal cell types in vitro, and implanted in vivo in porous ceramic vehicles to assess their osteogenic capacity, using human MSCs and brain pericytes as controls. The differentiation assays showed a difference between adipose tissue-derived cells as compared to equine bone marrow MSCs. While differences in CFU-F frequencies between both donors were evident, the CFU-F numbers correlated directly with blood vessel densities (r(2) = 0.86). We consider these preliminary data as further evidence linking MSCs to blood vessels.

FIG. 1.

Osteogenic differentiation of equine bone marrow MSCs. Calcium-rich extracellular matrix was stained with Alizarin Red S after 3 weeks under osteogenic differentiation conditions. (A, B) Horse 1 and Horse 2 MSCs, respectively. (C) HBVPs. (D) hMSCs (1400-2). Color images available online at www.liebertonline.com/ten.

FIG. 2.

Chondrogenic differentiation of eATDCs. Chondrogenic pellets were generated from eATDCs from HV and PV sites from two equine donors (Horse 1 and Horse 2) or from hMSCs (1398-1). (A) GAG accumulation at 1, 2, and 3 weeks. (B) Histological sections from pellets harvested at 1, 2, and 3 weeks stained with Toluidine Blue. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Adipogenic differentiation of eATDCs. eATDCs from HV and PV sites or from bone marrow from two equine donors (Horse 1 and Horse 2) were subjected to adipogenic differentiation using hMSCs (1398-2) as a positive control. At the end of the differentiation period, cells were stained using Oil Red O. (A) Human cells. (B) eATDCs from Horse 1's HV site. (C) eATDCs from Horse 1's PV site. (D) Horse 1's bone marrow cells. (E) eATDCs from Horse 2's HV site not subjected to adipogenic differentiation. (F) eATDCs from Horse 2's HV site. (G) eATDCs from Horse 2's PV site. (H) Horse 2's bone marrow cells. No adipocytes could be observed in (E), (D), and (H). Color images available online at www.liebertonline.com/ten.

FIG. 4.

Correlation between CFU-F number/mg of tissue and blood vessel area. The mean number of CFU-Fs of each horse adipose tissue–derived cell sample was normalized using sample wet weights and as a function of the mean area occupied by blood vessels. Points corresponding to PV and HV samples from each equine donor (Horse 1 and Horse 2) are indicated. The correlation coefficient (r²) was calculated using Microsoft Excel. Error bars represent SEM.