br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments This work was supported by grants to W.H. from the National Natural Science Foundation of China (81372258, 81572827), the Major State Basic Research Development Program of China (2013CB910802), and the Program for Professor of Special Appointment (Eastern Scholar, GZ2014002) at Shanghai Institutions of Higher Learning.
Introduction The discovery of transplantable hematopoietic apigenin with stem cell properties in mice half a century ago (Siminovitch et al., 1963; Till and McCulloch, 1961; Wu et al., 1967) was rapidly translated into a clinical therapeutic modality. Transplants of human hematopoietic stem cell (HSC)-containing products now form a key component of curative treatments for many diseases (Thomas, 1993). New applications are becoming increasingly feasible due to the widening availability of cord blood (CB) units and advances in the genetic modification of human HSCs (Naldini, 2015). The field has been further galvanized by increasing evidence of early transforming events in human leukemogenesis that target HSCs (Fearon et al., 1986; Lindsley et al., 2015; Prchal et al., 1978; Shlush et al., 2014). In mice, it has been possible to show that individual HSCs with durable regenerative activity can be greatly expanded in vivo with lifetime retention of their original functional potential (Dykstra et al., 2007; Harrison, 1979; Iscove and Nawa, 1997; Keller et al., 1985). Years of persisting hematopoiesis in patients given gene-marked autologous cells (Aiuti et al., 2013; Biffi et al., 2013; Cartier et al., 2009; Cavazzana-Calvo et al., 2010) indicate human HSCs maintained ex vivo for a few days can also remain active for many years post-transplant. We have previously shown that the survival, proliferation, and maintenance of the regenerative potential of mouse HSCs able to produce serially transplantable progeny can be differentially and directly regulated ex vivo by different combinations of external cues (Wohrer et al., 2014). In contrast, a detailed analysis of the direct effects of similarly defined human HSCs to external factors has remained elusive. However, this situation has recently changed with the identification of the CD34+CD38−CD45RA−CD90+CD49f+ subset of human CB cells (hereafter referred to as CD49f+ cells) as a highly enriched source of HSCs with long-term repopulating potential in transplanted immunodeficient mice (∼10% purity) (Notta et al., 2011). Combinations of five human growth factors (GFs), i.e., stem cell factor (SCF), Flt3-ligand (FLT3L), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF), were previously shown to expand the number of primitive adult human hematopoietic cells identified in vitro as long-term culture-initiating cells when maintained in vitro for up to 10 days (Petzer et al., 1996a, 1996b, Zandstra et al., 1997, 1998). Subsequent experiments showed the same five-GF combination modestly expanded (2-fold) CB cells that could regenerate multi-lineage hematopoiesis for a few weeks in sublethally irradiated NOD/SCID mice in 7-day cultures (Conneally et al., 1997). We now report the differential effects of the same five GFs, analyzed alone and in various combinations on the survival, proliferation, and serial regenerative activity of purified human CD49f+ CB cells. The results establish the ability of the five-GF combination to promote every viable cell to divide while retaining serially transplantable human HSC numbers over a 4- to 21-day period in vitro. Additional single-cell tracking studies demonstrate that these GFs regulate the short-term (4 day) survival and proliferation of human HSCs directly in a tunable and combinatorial fashion, but independently of the maintenance of their long-term regenerative activity in vivo.
Results
Discussion
Experimental Procedures
Author Contributions
The authors thank Margaret Hale and Glenn Edin for technical assistance and the British Columbia Cancer Agency Stem Cell Assay Laboratory for assistance in the processing and cryopreservation of CB samples. This work was supported by grants to C.E. from the Terry Fox Foundation New Frontiers Program Project Grant, a grant from the Stem Cell Network of Centers of Excellence, and a grant from Collaborative Health Research Projects. D.J.H.F.K. was supported by a Canadian Institutes of Health Research (CIHR) Vanier Scholarship. P.H.M. was supported by a CIHR Frederick Banting and Charles Best Canada Doctoral Scholarship.