Differential bone marrow stem cell mobilization by G-CSF injection or arterial ligation in baboons

Abstract

Bone marrow stem cells (BMSCs) are mobilized in response to ischemic attacks, e.g. myocardial infarction, to repair the damage, or by cytokines, e.g. granulocyte colony-stimulating factor (G-CSF), which is used to harvest BMSCs for autologous transplantation. In order to optimize BMSC mobilization strategy for cardiovascular repair, we investigated whether BMSCs mobilized by G-CSF share the same subtype profile as that by ischemia in a non-human primate model. We subjected five baboons to subcutaneous G-CSF injection and five baboons to femoral artery ligation. Blood BMSCs were measured by surface antigens; functional differentiation to endothelial cells (ECs) was assessed by colony-forming capacity, expression of mature EC antigens and tube-like formation. The number of circulating CD34+/CD45RA- cells spiked on day 3 post-stimulation in both groups. While the number of CD34+ cells released by artery ligation was 2-fold lower by comparison with the number released by G-CSF administration, significantly more CD133+/KDR+/CXCR4+/CD31+ cells were detected in the baboons that underwent artery ligation. After culture in endothelial growth medium, mononuclear cells from baboons with artery ligation formed more EC colonies and more capillary-like tubes (P < 0.05), expressed higher vWF and phagocytosed more Dil-Ac-LDL (P < 0.05). While G-CSF and artery ligation can mobilize BMSCs capable of differentiating into ECs, BMSCs mobilized by the artery ligation simulating in vivo ischemic attacks have higher potential for vascular differentiation. Our findings demonstrate that different mobilization forces release different sets of BMSCs that may have different capacity for cardiovascular differentiation.

Figure 1

Time‐dependent changes in circulating CD34+ cells mobilized by femoral artery ligation or G‐CSF administration. Freshly isolated PBMNCs were quantitatively evaluated by FACS analysis. (A) A representative FACS analysis including the gated CD34/CD45RA (A1), isotope control (A2), and a typical result for CD34+/CD45RA− (A3). (B) Time‐dependent changes of circulating CD34+/CD45RA− (B1) and CD34+/CD45RA+ (B2) cells in peripheral blood after femoral artery ligation (gray) and administration of G‐CSF (black). Due to veterinary restriction, no blood was collected in 24 hrs after the femoral artery ligation. Results are expressed as mean ± S.D., and five baboons were studied in each group.

Figure 2

Colony‐forming capacity of the mobilized PBMNCs. Total endothelial progenitor cell colony‐forming units (EPC‐CFU) and haematopoietic stem cell colony‐forming units (HSC‐CFU including EB‐CFU and GM‐CFU) from PBMNCs of baboons that received different treatments were counted at three time‐points. Typical examples of the colonies are shown as follows. (A) An EPC colony that consisted of a central core of rounded cells surrounded by sprouting cells. (B) A granulocyte macrophage (GM) colony, a type of HSC colony, characterized by its unique morphology of a population of round cells with no sprouting cells at the periphery. (C) The other type of the HSC colony is represented by erythroid burst (EB) colony featured by a group of cells with various sizes and with strong red‐colour appearance. The numbers of these colonies were counted and the results are expressed as mean ± SD (N= 5). Statistical significance was evaluated using the paired Student’s t‐test for comparisons between different time‐points and baseline levels *P < 0.05 and **P < 0.01; ^† P < 0.05, ^†† P < 0.01 for comparisons between days 3 and 10.

Figure 3

Endothelial‐specific differentiation of PBMNCs mobilized by femoral artery ligation or G‐CSF administration. PBMNCs collected on day 3 from both groups were cultured for 14 days before being labeled with Dil‐Ac‐LDL. (A) The average Dil‐Ac‐LDL uptakes assessed using CellQuest software are shown in baboons with artery ligation (light line, n= 5) and with G‐CSF administration (bold line, n= 5), along with the cells incubated with vehicles (dashed line, n= 10). To confirm the uptake of Dil‐Ac‐LDL, fluorescence microscopy at 600× magnification was carried out; a representative image is presented (bottom panel). (B) A representative FACS histogram for the binding of UEA‐1 binding on PBMNCs after 14 days of culture from baboons with artery ligation (B1) and baboons with G‐CSF administration (B2). Mature ECs as positive control (B3). Cells were cultured for 14 days before being labeled with UEA‐1 conjugated with FITC label (right column) or with vehicle (left column).

Figure 4

Direct observation of tube‐forming process by BMMNCs cultured on fibronectin‐coated plates. BMMNCs were initially suspended in angiogenic culture conditions and plated in fibronectin‐coated wells. After 7 days of culture, some cells with oval or spindle shapes aggregated together, as indicated by arrow (A, 100× magnification). After 4 weeks of culture, cells were differentiated further and two tube‐like structures were seen: a complex cord‐like structure with either two layers of cells (B, 200× magnification) or multiple layers (C and D, 100× magnification). Some of the tubes also formed a branching point, which represents a capillary‐like structure (E, 100× magnification). Panel (F) shows the capillary‐forming structures at higher magnification (400×). Panel (G) shows the capillary‐like structure formed by BMMNCs grown in Matrigel. A wide range of sizes were consistently seen in cultures from baboons with artery ligation by comparison with the G‐CSF treated group. Three circular capillary‐like structures are indicated by arrows (400×). In contrast, more cell aggregates or clusters (H) were presented in PBMNCs collected from baboons treated with G‐CSF than with artery ligation.