Poor functional recovery after transplantation of diabetic bone marrow stem cells in ischemic myocardium

Abstract

Background: Autologous bone marrow mononuclear cell (BMMC) therapy has shown promise for improving cardiac function after myocardial infarction. The efficiency of such therapy for diabetic patients remains unknown.

Methods: BMMCs were harvested from type 2 diabetic male BKS.Cg-m+/+Lepr(db)/J mice or C57BLKS/J (non-diabetic control) mice and were isolated using Ficoll-based separation. Cell characterization was performed by flow cytometry. Cell viability was determined by apoptosis and proliferation assays. Female BKS.Cg-m+/+Lepr(db)/J mice underwent left anterior descending artery ligation and were randomized into 3 groups receiving 2.5 x 10(6) diabetic BMMCs (n = 8), 2.5 x 10(6) control BMMCs (n = 8), or phosphate-buffered saline (n = 6). At Week 5, cardiac function was assessed with echocardiography and invasive hemodynamic measurements. Post-mortem cell survival was quantified by TaqMan real-time transcription polymerase chain reaction (RT-PCR) for the male Sry gene.

Results: BKS.Cg-m+/+Lepr(db)/J BMMCs showed a significantly lower mononuclear fraction and a significantly lower proliferation rate compared with C57BLKS/J BMMCs. Fractional shorting (40.1% +/- 1.2% vs 30.3% +/- 1.9%; p = 0.001) and cardiac output (4,166 +/- 393 vs 2,246 +/- 462 microl/min; p = 0.016) significantly improved for mice treated with control BMMCs injection compared with those treated with diabetic BMMCs, respectively. This difference could not be attributed to difference in cell engraftment because TaqMan RT-PCR showed no significant difference in cell survival in infarcted hearts between the 2 groups.

Conclusions: Diabetic BMMCs are significantly impaired in their ability to improve cardiac function after myocardial infarction compared with control BMMCs. These findings could have significant clinical implication regarding autologous BMMC therapy in diabetic patients.

Figure 1

Characterization of bone marrow mononuclear cells. (A) The identity of BMMC subpopulation was investigated by fluorescent-activated cell sorting (FACS) with antibodies recognizing Sca-1, Mac-1, GR-1, CD45, CD34, and CD133. (B) After Ficoll gradient (an isolation procedure as seen in clinical trials), the db/db group has significantly lower mononuclear (MN) cell fraction and Sca-1 population but higher Mac-1¹⁺/Gr-1⁺ fraction. This indicates that the db/db BMMC injections used have a significantly lower fraction of MN cells than the control BMMC injections. Moreover, within the db/db group there is a higher contamination of granulocytes. No significant differences in CD45^negCD34⁺ or CD45^negCD133⁺ were found (data not shown). Graphs represent 3 independent experiments. Abbreviations: BMMCs, bone marrow mononuclear cells.

Figure 2

In vitro cell viability and proliferation assays. (A) Apoptosis measured after 24 hours of incubation in either normoxic conditions or hypoxic conditions. BMMCs from control or db/db donors showed the same trend in normoxia. (B) For proliferation capacity, BMMCs were cultured for 36 hours under normoxic and hypoxic conditions and cell quantitation was performed with the MTT assay. After 36 hours, db/db BMMCs showed a significantly impaired proliferation rate as compared to control BMMCs under both normoxic conditions and hypoxic conditions. Data are expressed as percentage relative to 100% at t=0 (*P <0.05). Abbreviations: MTT assay, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Figure 3

Left ventricular function following BMMC therapy. (A) Representative M-mode images of heart at 5 weeks after LAD ligation with administration of control BMMCs (left), db/db BMMCs (middle), or PBS control (right). (B) Quantification of left ventricular fractional shortening (LVFS) demonstrated a significant improvement at 5 weeks post-MI in animals receiving control BMMCs compared to db/db BMMCs or PBS (*P < 0.05). Abbreviations: M-mode, motion mode; PBS, phosphate-buffered saline; LAD, left anterior descending.

Figure 4

Invasive hemodynamic measurements using pressure-volume loops. (A) At day 35, cardiac output was significantly increased in hearts receiving control BMMCs compared to hearts receiving db/db BMMCs or PBS (*P<0.05). (B) Moreover, arterial elastance was significantly decreased in hearts receiving control BMMCs compared with hearts receiving PBS (*P<0.05). (C–F) At day 35, there was a trend toward improved functional recovery in animals receiving control BMMCs compared with animals receiving db/db BMMCs or PBS. This trend did not achieve statistical significance compared with db/db BMMCs.

Figure 5

Real-time polymerase chain reaction quantification of surviving male transplanted BMMCs within female heart. Ex vivo Taqman analysis of hearts undergoing LAD ligation following injection of db/db BMMCs versus control BMMCs revealed no significant difference in survival of transplanted cells. In both hearts cell, survival at day 35 after surgery was <1% (n=3 in control and n=5 in db/db group).