Presentation Title

In Vitro Expansion of Hematopoietic Stem and Progenitor Cells Using Small Molecules and Human Umbilical Vein Endothelial Cell Co-Cultures

Start Date

November 2016

End Date

November 2016

Location

HUB 302-76

Type of Presentation

Poster

Abstract

Hematopoietic stem cells (HSCs), which can differentiate into mature blood cells and self-renew, have been transplanted to treat hematologic disorders. However, HSC numbers are a limiting factor to transplant success. Studies report various methods for HSC expansion in vitro, but each study definition of the HSC phenotype differs, precluding reliable translation into clinical practice.1,2,3,4 Our groups have identified an evolutionarily conserved HSC phenotype (CD34+, CD133+, CD45RA-, CD90+) that correlates directly with engraftment potential post transplantation in nonhuman primate and xenotransplantation models.5,6,7 Here we evaluated four small molecules for HSC expansion (University of Montreal molecule 171 [UM171], StemRegenin 1 [SR1], Ly2228820, and Prostaglandin 2 [PGE2]), as well as endothelial cell (EC) co-culture to expand our HSC phenotype. Cryopreserved CD34+ cells from three individual healthy donor mobilized apheresis products were cultured for 7 days in each of the expansion protocols. Cells were counted and analyzed by flow cytometry for HSC phenotype. Differentiation potential of expanded and non-expanded cells was compared by colony forming cell (CFC) assays. We determined that 40nM UM171 alone, and in combination with both SR1 and Ly, significantly expanded HSCs ~3-fold over other conditions (p ≤ 0.02). UM171-expanded HSCs also showed the greatest differentiation potential in CFC assays compared to other expansion conditions (p ≤ 0.05). Thus, UM171 alone or combined with SR1 and Ly shows the most promise for in vivo testing in murine transplantation studies. Further characterization may help to define mechanisms of HSC maintenance and proliferation beneficial to improving HSC treatments including gene therapy.

References

(1) Fares, I et al. (2014) Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal, Science, 345, 1509-1512.

(2) Tesio, M et al. (2015) Hematopoietic stem cell quiescence and function are controlled by the CYLD–TRAF2–p38MAPK pathway, JEM, 212, 525-538.

(3) Hagedorn, E et al. (2014) Getting more for your marrow: boosting hematopoietic stem cell numbers with PGE2, Exp Cell Res, 329, 220-226.

(4) Raynaud, C et al. (2013) Endothelial cells provide a niche for placental hematopoietic stem/progenitor cell expansion through broad transcriptomic modification, Stem Cell Res, 11, 1074-1090.

(5) Radtke, S et al. (2015) CD133 allows elaborated discrimination and quantification of haematopoietic progenitor subsets in human haematopoietic stem cell transplants, Br J Haematol, 169, 868-878.

(6) Additional Radtke et al. manuscript submitted.

(7) Patent application 62/351,761.

This document is currently not available here.

Share

COinS
 
Nov 12th, 1:00 PM Nov 12th, 2:00 PM

In Vitro Expansion of Hematopoietic Stem and Progenitor Cells Using Small Molecules and Human Umbilical Vein Endothelial Cell Co-Cultures

HUB 302-76

Hematopoietic stem cells (HSCs), which can differentiate into mature blood cells and self-renew, have been transplanted to treat hematologic disorders. However, HSC numbers are a limiting factor to transplant success. Studies report various methods for HSC expansion in vitro, but each study definition of the HSC phenotype differs, precluding reliable translation into clinical practice.1,2,3,4 Our groups have identified an evolutionarily conserved HSC phenotype (CD34+, CD133+, CD45RA-, CD90+) that correlates directly with engraftment potential post transplantation in nonhuman primate and xenotransplantation models.5,6,7 Here we evaluated four small molecules for HSC expansion (University of Montreal molecule 171 [UM171], StemRegenin 1 [SR1], Ly2228820, and Prostaglandin 2 [PGE2]), as well as endothelial cell (EC) co-culture to expand our HSC phenotype. Cryopreserved CD34+ cells from three individual healthy donor mobilized apheresis products were cultured for 7 days in each of the expansion protocols. Cells were counted and analyzed by flow cytometry for HSC phenotype. Differentiation potential of expanded and non-expanded cells was compared by colony forming cell (CFC) assays. We determined that 40nM UM171 alone, and in combination with both SR1 and Ly, significantly expanded HSCs ~3-fold over other conditions (p ≤ 0.02). UM171-expanded HSCs also showed the greatest differentiation potential in CFC assays compared to other expansion conditions (p ≤ 0.05). Thus, UM171 alone or combined with SR1 and Ly shows the most promise for in vivo testing in murine transplantation studies. Further characterization may help to define mechanisms of HSC maintenance and proliferation beneficial to improving HSC treatments including gene therapy.

References

(1) Fares, I et al. (2014) Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal, Science, 345, 1509-1512.

(2) Tesio, M et al. (2015) Hematopoietic stem cell quiescence and function are controlled by the CYLD–TRAF2–p38MAPK pathway, JEM, 212, 525-538.

(3) Hagedorn, E et al. (2014) Getting more for your marrow: boosting hematopoietic stem cell numbers with PGE2, Exp Cell Res, 329, 220-226.

(4) Raynaud, C et al. (2013) Endothelial cells provide a niche for placental hematopoietic stem/progenitor cell expansion through broad transcriptomic modification, Stem Cell Res, 11, 1074-1090.

(5) Radtke, S et al. (2015) CD133 allows elaborated discrimination and quantification of haematopoietic progenitor subsets in human haematopoietic stem cell transplants, Br J Haematol, 169, 868-878.

(6) Additional Radtke et al. manuscript submitted.

(7) Patent application 62/351,761.