Maitake beta-glucan enhances umbilical cord blood stem cell transplantation in the NOD/SCID mouse

Abstract

Beta glucans are cell wall constituents of yeast, fungi and bacteria, as well as mushrooms and barley. Glucans are not expressed on mammalian cells and are recognized as pathogen-associated molecular patterns (PAMPS) by pattern recognition receptors (PRR). Beta glucans have potential activity as biological response modifiers for hematopoiesis and enhancement of bone marrow recovery after injury. We have reported that Maitake beta glucan (MBG) enhanced mouse bone marrow (BMC) and human umbilical cord blood (CB) cell granulocyte-monocyte colony forming unit (GM-CFU) activity in vitro and protected GM-CFU forming stem cells from doxorubicin (DOX) toxicity. The objective of this study was to determine the effects of MBG on expansion of phenotypically distinct subpopulations of progenitor and stem cells in CB from full-term infants cultured ex vivo and on homing and engraftment in vivo in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse. MBG promoted a greater expansion of CD34+CD33+CD38- human committed hematopoietic progenitor (HPC) cells compared to the conventional stem cell culture medium (P = 0.002 by ANOVA). CD34+CXCR4+CD38- early, uncommitted human hematopoietic stem cell (HSC) numbers showed a trend towards increase in response to MBG. The fate of CD34+ enriched CB cells after injection into the sublethally irradiated NOS/SCID mouse was evaluated after retrieval of xenografted human CB from marrow and spleen by flow cytometric analysis. Oral administration of MBG to recipient NOS/SCID mice led to enhanced homing at 3 days and engraftment at 6 days in mouse bone marrow (P = 0.002 and P = 0.0005, respectively) compared to control mice. More CD34+ human CB cells were also retrieved from mouse spleen in MBG treated mice at 6 days after transplantation. The studies suggest that MBG promotes hematopoiesis through effects on CD34+ progenitor cell expansion ex vivo and when given to the transplant recipient could enhance CD34+ precursor cell homing and support engraftment.

Figure 1

Effect of MBG on expansion of HPC and HSC in cord blood ex vivo. Panel A. Mononuclear cells were separated from cord blood from healthy infants (n = 4) enriched for CD34+ cells and cultured ex vivo in the presence or absence of MBG at the indicated doses. The effects of MBG on expansion of cell populations were determined after 4 days of culture followed by harvesting, staining with anti-human CD34, CD38, CD33 antibodies, and assessment by three-color flow cytometry. Data in Panel A show the CD34+CD33+D38- (HPC) cell population. Data in Panel B show the CD34+CD38-CXCR4+ (HSC) cell population from the same cultures as in Panel A. Data present mean cell numbers ± SD, * P< 0.05 vs. control with no MBG.

Figure 2

Expression of dectin-1 on cord blood cells. CB samples were stained with dectin-1 antibody, using a modified indirect staining protocol to detect dectin-1, and directly conjugated fluorescent antibodies to CD45, CD19, and anti-CD14 were used for assessment of B cells and monocytes; all analyzed by three-color flow cytometry. Data in Panel A show flow cytometric histogram overlays for one-term infant’s monocyte and B cell populations. Data show the percentage of each respective gated population that expresses dectin-1 compared to the isotype control. Data in Panel B represent the mean percentage ± SD of monocytes and B cells in the CB group that express dectin-1. Samples were from 12 healthy full-term infants.

Figure 3

Effect of MBG on homing of CB CD34+ cells in NOD/SCID mice. Data show effect of daily oral MBG treatment at 4 mg/kg/day at 3 days after CB transplant compared to control group mice. Control 1 (Ctr1) group mice (n = 4) were transplanted with same CB as the MBG1 group of mice (n = 4), while control 2 (Ctr2) mice (n = 4) and MBG 2 mice (n = 4) received the same other unit of CB. CD34+CD45+ human CB cells were retrieved from bone marrow (A) and spleen (B) and analyzed by flow cytometry (** P < 0.01 vs. control).

Figure 4

Effects of MBG on engraftment of CB CD34⁺ cells in NOD/ SCID mice. The MBG group mice were given 4 mg/kg/day of MBG beginning on the day of CB transplantation and during the subsequent 6 days. Control 1 (Ctr1) group of mice and MBG1 group mice were transplanted with the same unit of CB, while control 2 (Ctr2) group of mice and the MBG2 group of mice received the other same unit of CB. Human CD34+CD45+ cells retrieved from NOD/ SCID mice bone marrow (A) or spleen (B) were analyzed by flow cytometry (** P < 0.01 vs. control).

Figure 5

Comparison of response to MBG after transplantation in NOD/SCID mice. The MBG group mice were given 4 mg/kg/day of MBG on the day of transplantation and over the subsequent 3 or 6 days (n = 8, n = 8, respectively). Human CD34+CD45+cells retrieved from NOD/SCID mice bone marrow (Panel A) or spleen (Panel B) were analyzed by flow cytometry. Data show mean percentage ± SD, ** P < 0.01 vs. control.