Flow cytometric analysis of bone marrow-derived CD45RA-positive B cells revealed that the injected population of CD45RA selected B cells were relatively free of other lineage-positive bone marrow cells including cytotoxic T cells (CD8+) (B), natural killer T cells (CD3) (C), helper T cells (CD4) (D), and neutrophils (E). Isotype control is shown in A. Flow cytometric analysis of bone marrow-derived CD45RA-positive B cells confirmed the majority of the B cells are CD45R-positive lymphocytes (G). IgM-positive immature B cells (H), IgG-positive mature B cells (I), and a minority of early c-kit⁺ positive pro-B cells (L). The bone marrow-derived B cell fraction did not contain CD5-positive marginal cells (J). Isotype control for CD45R, IgM, IgG, and CD5 antibodies is shown in F, and isotype control for c-kit is shown in K. Representative flow cytometry data are shown.

Two weeks after transplantation of B lineage and stem cell populations into infarcted rat myocardium, the surviving animals underwent follow-up echocardiographic assessment. Representative M-mode echocardiography is shown in A. B is a graph of normal (shaded squares, n = 9), immediate post-ligation (shaded circles, n = 9), sham operated (no ligation, open squares, n = 15), and ligation plus saline injection (open circles, n = 15) animals were included as control animals. Solid triangles represent animals injected with hematopoietic stem cells (HSC) (n = 4); solid diamonds represent animals injected with HSC coinjected with B cells (n = 5); solid circles represent B cell-injected animals (n = 24). B cells cultured overnight at 37°C (n = 11) are represented by solid squares, and B cells cocultured with nonhematopoietic stem cells (NHSCs) cultured overnight at 37°C are represented by patterned squares (n = 18). Each data point represents 1 animal, and horizontal bars are the mean ± SE. Significant differences in the mean values are denoted by *p < 0.05 using analysis of variance with post-hoc analysis using Fisher least significant difference.

Animals were implanted with a 5-bromo-2'-deoxyuridine pellet at the time of infarction and cell injection for determination of cumulative cellular proliferation. After 2 weeks, animals were euthanized and hearts processed for assessment of 5-bromo-2'-deoxyuridine pellet incorporation into the nuclear DNA of proliferating cells of ligation plus saline-treated animals (A), or B cells cocultured with nonhematopoietic stem cells (B), B cell (C) and cultured B cell (D) injections. Positive cells were identified using antibodies directed against a 5-bromo-2'-deoxyuridine pellet. All nuclei were stained with hematoxylin. The percentage of cells staining positive for 5-bromo-2'-deoxyuridine pellet was determined by comparison with the total number of cells visualized in a minimum of 5 fields (200x magnification). Scale bars are 100 µm. Sham operated (no ligation, open squares, n = 5) and ligation plus saline injection (open circles, n = 6) animals were included as control animals (E). Solid circles represent animals injected with B cells (n = 10). B cells cultured overnight at 37°C (n = 10) are represented by solid squares, and B cells cocultured with nonhematopoietic stem cells cultured overnight at 37°C (n = 4) are represented by patterned squares. Each data point represents the percentage of 5-bromo-2'-deoxyuridine-positive cells counted in a single field, and the horizontal bar represents the mean ± SE. Significant differences in mean values between control and experimental groups are denoted by *p < 0.05 using the Student t test, 2-tailed.

Double immunohistochemical detection of proliferating 5-bromo-2'-deoxyuridine-positive cells with von Willebrand factor-positive endothelial cells (A to D) and cardiac myocytes (E to H) within the border zone of rat infarcted myocardium was determined 2 weeks after transplantation of saline control cells (A and E), B cells (B and F), cultured B cells (C and G), and B cells cocultured with nonhematopoietic stem cells (D and H). Positive 5-bromo-2'-deoxyuridine-labeled cells were visualized using 3, 3'-diaminobenzidine in which all positive cells are stained brown and von Willebrand factor and sarcomeric actin staining was visualized using Vector red. Double positive staining is denoted by white arrows. All nuclei were stained with hematoxylin. Instrument magnification 400x. Scale bars are 50 µm.

Histological assessment of cellular apoptosis was performed using the TUNEL assay. Animals underwent sham (no ligation) (A) operation or ligation with B cell (B) or B cell cocultured with nonhematopoietic stem cell (NHSC) (C) injections. Animals were euthanized at 48 h after surgery, and hearts were processed for evaluation of apoptosis. Apoptotic cells within the peri-infarct domain were quantitated by visualization using 200x magnification. Scale bars are 100 µm. All nuclei were stained with hematoxylin. The percentage of cells staining positive for apoptosis was determined by comparison with the total number of cells visualized in a minimum of 5 fields. Ligation plus saline injection (open circles, n = 6) animals were included as control animals (D). Solid circles represent animals injected with B cells (n = 6), and B cells cocultured with NHSCs cultured overnight at 37°C (n = 4) are represented by solid squares. Each data point represents the percentage of TUNEL-positive cells counted in a single field in cardiac section with the largest infarction and horizontal bar is the mean ± SE. Significant differences in the mean values between control and experimental groups are denoted by *p < 0.05 using the Student t test, 2-tailed.

Immunohistochemical detection of CD45RA-positive cells within the border zone of rat infarcted myocardium 2 weeks after transplantation of saline control cells (A), B cells (B), cultured B cells (C), and B cells cocultured with nonhematopoietic stem cell (D). Tissue sections were stained with rat-specific antibody against CD45RA and visualized using 3, 3'-diaminobenzidine in which all positive cells are stained brown (arrows). All nuclei were stained with hematoxylin. Instrument magnification 400x. Scale bars are 50 µm.