Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects

Abstract

The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

Figure 1

Stem-Kine Supplementation Augments Circulating CD133 Expressing Cells. PBMC from 18 healthy volunteers were assessed by flow cytometry for expression of CD133 at days 0, 1, 2, 7, and 14 after initiation of twice daily Stem-Kine administration. Data is presented as percentage over control of average values from all 18 subjects. *P < 0.05 compared to pre-treatment group.

Figure 2

Stem-Kine Supplementation Augments Circulating CD34 Expressing Cells. PBMC from 18 healthy volunteers were assessed by flow cytometry for expression of CD34 at days 0, 1, 2, 7, and 14 after initiation of twice daily Stem-Kine administration. Data is presented as percentage over control of average values from all 18 subjects. *P < 0.05 compared to pre-treatment group.

Figure 3

Stimulation of Hematopoietic Progeny from PBMC (HALO Assay): PBMC were plated at a concentration of 20,000 cells per well and cultured on a methylcellulose matrix for 5 days supplemented with; (a) control media or (b) an optimized hematopoietic growth factor cocktail as described in Materials and Methods.

Figure 4

Stem-Kine Supplementation Increases Hematopoietic Progenitor Cells in Circulation. PBMC from subjects supplement with Stem-Kine were extracted at the indicated timepoints and cultured for 5 days in the presence of control media or hematopoietic cytokines. Ratio of ATP between activated and control cells is illustrated on the y-axis. *P < 0.05 compared to pre-treatment groups.

Figure 5

Augmentation of KDR/CD34 positive cell numbers in circulation after Stem-Kine administration. PBMC from 18 healthy volunteers were assessed by flow cytometry for coexpression of CD34 and KDR at days 0, 1, 2, 7, and 14 after initiation of twice daily Stem-Kine administration. *P < 0.05 compared to pre-treatment groups.

Figure 6

Colony Forming Unit Endothelium Assay: PBMC were plated on 24-well fibronectin-coated plates at a concentration of 10(6) cells per well. After 5 days of culture cells were Giemsa stained and clusters of > 50 cells were quantified as colonies.

Figure 7

Stem-Kine Supplementation Augments Circulating Cells with CFU-E Generating Activity. CFU-E were generated by incubation of PBMC isolated from healthy volunteers with EndoCult Media. Data is presented as ratio to pre-treatment values. Open squares represent quantification by Alpha-Ease software, whereas closed symbols indicate quantification per viewing field by microscope. *P < 0.05 compared to pre-treatment groups.