Spleens and sections from livers, lungs, and kidneys were processed in parallel. 5 ��m frozen sections were mounted on Superfrost microscope slides. Human cells were detected using human specific antibodies: rabbit anti-��2-Microglobulin (1:800, Abcam, Cambridge, United Kingdom), mouse anti-CD45 (1:200, Vector Laboratories, Burlingame, CA) and mouse anti-CD31 (1:100, DAKO, Glostrup, but Denmark). Staining was visualized using highly cross-adsorbed goat anti-mouse or anti-rabbit secondary antibodies conjugated with either Alexa488 or Alexa594 antibodies (1:000, all Invitrogen) and sections were mounted with DAPI containing Neomount mounting medium (Invitrogen). Relevant isotype controls were stained in parallel. Comparable frozen sections of hearts from PBS injected mice or human heart were used as negative and positive controls, respectively.

Sections were analyzed on a Zeiss Axiovert4000 wide field fluorescent microscope (Carl Zeiss Inc., Oberkochen, Germany) using the Metamorph software (Molecular Devices, Sunnyvale, CA). Image stacks of thin serial sections were obtained from selected sections by Z-stage scanning. Blinded 3D deconvolution (Autoquant, Media Cybernetics, Inc., MD) was used to reduce out of focus light and enhance signal to noise ratio. Single thin optical sections were generated using the ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2006).

Vascular density 5 ��m frozen sections from the basal and medial portion of the hearts from each treatment group (PBS: n = 12; ALDHloLin-: n = 5; ALDHhiLin-: n = 9) were stained with mouse-specific rat anti-CD31 antibody (1:100, BD Biosciences, San Diego, CA) and visualized using a HRP-conjugated secondary goat anti-mouse antibody (Acriz Antibodies GmbH, Hiddenhausen, Germany) and DAB+ chromagen according to the manufacturer’s instruction (DAKO). For each heart, bright field images were recorded from 10 randomly selected visual fields (40�� magnification) in the tissue sub-served by the infarct related artery. Mean vascular density per ��m2 tissue was estimated for each group. Only CD31 positive structures with a well defined tubular morphology or structures with a linear extension equal to or larger than 50 ��m were scored as positive. Images were analyzed using the ImageJ software.

Statistical analyses All data were analyzed by ANOVA with Bonferroni correction for multiple comparisons. p-values smaller than or equal to 0.05 were considered significant. Hadis method AV-951 to identify outliers in multivariate data[21] was applied to the vascular density data with a 95% significance level. Results Distribution of ALDHloLin-, ALDHhiLin-, and CD34+ cells at 48-72 hours post transplantation We first evaluated the short term homing potential of three human stem and progenitor cell populations, ALDHhiLin-, ALDHloLin-, and CD34+, purified from UCB as previously described[8].