In vitro protection of umbilical cord blood–derived primitive hematopoietic stem progenitor cell pool by mannose-specific lectins via antioxidant mechanisms
Ashwini S. Hinge
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorLalita S. Limaye
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorAvadhesha Surolia
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorVaijayanti P. Kale
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorAshwini S. Hinge
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorLalita S. Limaye
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorAvadhesha Surolia
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorVaijayanti P. Kale
From the National Center for Cell Science, Ganeshkhind, University of Pune Campus, Pune; and the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Search for more papers by this authorAH carried out all the experiments and wrote the manuscript; LSL is the co-investigator of the project and participated in the writing of the manuscript; AS isolated, purified, and characterized the lectins; and VPK is the principal investigator of the project, conceived the experiments, and wrote and edited the manuscript.
Abstract
BACKGROUND: Earlier we reported that an oral administration of two mannose-specific dietary lectins, banana lectin (BL) and garlic lectin (GL), led to an enhancement of hematopoietic stem and progenitor cell (HSPC) pool in mice.
STUDY DESIGN AND METHODS: Cord blood–derived CD34+ HSPCs were incubated with BL, GL, Dolichos lectin (DL), or artocarpin lectin (AL) for various time periods in a serum- and growth factor–free medium and were subjected to various functional assays. Reactive oxygen species (ROS) levels were detected by using DCHFDA method. Cell fractionation was carried out using lectin-coupled paramagnetic beads.
RESULTS: CD34+ cells incubated with the lectins for 10 days gave rise to a significantly higher number of colonies compared to the controls, indicating that all four lectins possessed the capacity to protect HSPCs in vitro. Comparative analyses showed that the protective ability of BL and GL was better than AL and DL and, therefore, further experiments were carried out with them. The output of long-term culture-initiating cell (LTC-IC) and extended LTC-IC assays indicated that both BL and GL protected primitive stem cells up to 30 days. The cells incubated with BL or GL showed a substantial reduction in the ROS levels, indicating that these lectins protect the HSPCs via antioxidant mechanisms. The mononuclear cell fraction isolated by lectin-coupled beads got enriched for primitive HSPCs, as reflected in the output of phenotypic and functional assays.
CONCLUSION: The data show that both BL and GL protect the primitive HSPCs in vitro and may also serve as cost-effective HSPC enrichment tools.
Supporting Information
Fig. S1. CD34+ cells were incubated with BL, GL, AL or DL for 10 days (100 pg/mL) and were subjected to CFU assays. A differential colony count was taken by scoring for the colonies belonging to various types. An increase in all types of colonies was seen in the lectin-treated cells indicating that the salutary effect of lectins was not restricted to any particular lineage.
Fig. S2. CD34+ cells were incubated with DL or AL (100 pg/mL) in presence or absence of alpha methyl mannoside sugar (20 mM) for 10 days and were subjected to CFU assay. Presence of the competing sugar abrogated the protective effect of lectins indicating the specific action of the lectins.
Fig. S3. CD34+ cells isolated from fresh cord blood samples were subjected to CFU assay as described above. 2 × 105 CD34+ cells from the same samples were incubated either with BL or GL (100 pg/mL) for 10 days and were subjected to CFU assay. A significantly more number of colonies were formed in the cells incubated with the lectins as compared to the control cells that were incubated without the lectins. There was no statistical difference in the number of CFU formed from freshly isolated cells versus the lectin-incubated cells, clearly indicating that the lectins maintain the freshness of the HSPCs (NS = Non Significant).
Fig. S4. CD34+ cells were incubated with either BL or GL for 10, 20 and 30 days. The cells were harvested after each time interval and the viable cell count was taken by trypan blue dye exclusion method. A significantly higher number of cells were present in the lectin-incubated cells as compared to the control indicating that the lectins exerted a protective effect on them.
Fig. S5. The surface of plates was coated with the lectins (10 ng/mL) in PBS at 37°C for 2 hours and 2 × 105 CD34+ cells were seeded per well in Stem Pro medium. Cells incubated in the uncoated wells were used as controls. After 10 days of incubation, the cells were harvested and were subjected to CFU assay. Significantly more numbers of colonies were observed in the cells incubated on the lectin-coated surfaces than the controls indicating that the lectins exert HSPC protective effect in an immobilized form as well.
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