Principal Investigator Harvey Lodish
No single known growth factor or combination of growth factors supported HSC expansion in culture. Furthermore existing lines of “supportive stromal cells” did not support expansion of HSCs; at best they maintained the level of HSCs over time, presumably due to a steady state between generation of new HSCs by division and differentiation of “old” stem cells. Thus, several years ago Chengcheng (Alec) Zhang turned to mouse fetal liver since the number of fetal HSCs normally increased markedly between embryonic Day 14 and Day 21. Chengcheng hypothesized that unknown growth proteins are produced by as- yet unidentified populations of fetal liver cells that stimulate the expansion of fetal liver HSCs. He identified Embryonic Day 15 fetal liver CD3+ Ter119- cells as a novel cell population that supports a net expansion of HSC numbers in culture. By transcriptional profiling of these cells and several others that do not support HSC expansion, Chengcheng uncovered several novel growth factors that, together, supported an unprecedented extent of ex vivo expansion of bone marrow HSCs: insulin - like growth factor 2 (IGF - 2) and Angiopoietin-like 2 and 3. A serum- free medium containing only low levels of stem cell factor (SCF), thrombopoietin (TPO), IGF-2, FGF-1, and Angiopoietin-like 2 or 3 stimulated a 24-30-fold expansion of HSCs following 10 days of culture of highly enriched mouse stem cells.
Next we found a ~20-fold expansion of human cord blood-derived CD133+ HSCs in a 10-day culture with a similar defined medium containing SCF, IGFBP2, TPO, FGF-1, and Angiopoietin-like 5, as measured by transplantation into NOD- scid immune- defective mice. In collaboration with Dr. Adam Drake and colleagues in Professor Jianzhu Chen’s lab at the MIT Koch Institute for Integrative Cancer Research, we used NOD-scid Il2rg-/- (NSG) mice that support long-term human HSC engraftment as recipients, and have definitively assessed the presence of HSCs in the expanded cell population. We showed that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells. The expanded cells also mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, the expanded CD34+ CD133+ cells efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in neonate recipients reconstituted with uncultured human HSCs. These findings suggest that these growth factors, Angiopoietin like 5, insulin growth factor binding protein 2, stem cell factor, thrombopoietin, and fibroblast growth factor 1, in the defined medium support the expansion of long-term human HSCs. The ability to expand human HSCs in vitro should facilitate clinical application of HSCs and large-scale construction of humanized mice from the same source for research applications. We are also collaborating William Hwang at the Singapore General Hospital to carry out preclinical and eventually clinical studies on ex vivo cord blood HSC expansion.