Our objective was to determine how the

Our objective was to determine how the culturing of CD34+ nicotinic receptors under conditions that influence ARNT complex formation might affect the expansion of HSPCs. We hypothesized that the use of culture conditions that stabilize HIF-1α and antagonize the AhR protein would promote the expansion of the primitive HSC population and thereby enhance the engraftment capacity of the LT-HSC population.
Materials and methods
Results
Discussion Previous studies have found that the ARNT transcription factor plays an important role in the regulation of hematopoiesis (Lindsey and Papoutsakis, 2012; Shimba et al., 2003). In order to further investigate how ARNT complex formation might benefit the expansion of the CD34+ sub-populations, cord blood CD34+ cells were cultured under selected conditions. Hypoxia (Ivan et al., 2001; Takubo et al., 2010) has a dose related effect on promoting cellular quiescence that in turn promotes the maintenance of HSPCs (Cheng et al., 2000; Guitart et al., 2010) by affecting cell proliferation, self renewal and differentiation (Ivanovic, 2009; Ivanovic et al., 2002). However, 1% O2 is not suitable for the expansion HSCs due to the suppression of cell proliferation which results in a low final number of HSCs after culture. Similar results were obtained for cultures treated with a low concentration of DMOG (0.1μM) which is a mimic of 1% O2. A minimal loss of the CD34 marker in the 20% O2/DMOG samples suggests that since there is no direct relationship between CD34 expression and maintenance of self renewal, DMOG slows down the differentiation process that occurs at 20% O2 by stabilizing HIF-1α. The 20% O2/DMOG sample proliferated at a slow rate, similar to the 1% O2 sample and the sizes of the cell sub-populations were also comparable. This demonstrates that the addition of 0.1mM DMOG to culture medium at 20% O2 can be used to inhibit differentiation in a manner that is almost similar to a hypoxic environment of 1% O2. The total number of cells in all the sub-populations is greater in the presence of 20% O2/DMOG than 1% O2, which makes 20% O2/DMOG a better condition for expanding HSCs. Cells cultured under normoxic conditions (20% O2), where it is known that HIF-1α is degraded and AhR binds ARNT (Boitano et al., 2010; Ivanovic, 2009), proliferate at a high rate and only maintain a small proportion of quiescent cells. However, even though the percentage of primitive and progenitor cells is low, the total/absolute number is significantly higher than in samples cultured under hypoxia. To determine if the expansion of primitive HSCs can be improved by limiting ARNT/AhR complex formation, CD34+ cord blood cells were cultured with SR1, an antagonist of AhR (Boitano et al., 2010). Isolated CD34+ cells cultured in 20% O2 with SR1, where it is known that both HIF-1α and AhR are suppressed (Boitano et al., 2010; Ivanovic, 2009), proliferate at a high rate. We demonstrate that this occurs mainly in the SP cell compartment which suggests that the expansion of the primitive HSC population is favored. This was confirmed following engraftment of NSG mice that provided a 4.23 fold increase in LT-SRC frequency obtained with a cell dose after expansion of 13,430 cells. The final number of primitive HSCs obtained after culture was far greater than the other conditions, which indicates that primitive HSC expansion is favored when cells are cultured under normoxic conditions with SR1. Cells cultured in both DMOG (Ivanovic, 2009) and SR1 (Boitano et al., 2010) proliferate at a similar rate to cultures containing DMOG alone. However, the primitive stem cell population is larger than when cells are cultured in DMOG alone as demonstrated by the increased proportion of cells that have side population characteristics as well as cells with short and long-term CFU abilities. Culturing cells in DMOG or hypoxia with SR1 is useful to obtain a high percentage of primitive progenitor cells. The engraftment data support this finding where the short-term and long-term (LT-SRC frequency of 3.8) engraftment was similar to the condition where HIF-1α was not stabilized and an AhR antagonist was used (LT-SRC frequency of 4.23). This was, however, obtained with a much lower cell dose after expansion of 3770 cells. This indicates that the 1% O2/SR1 treatment increased the capacity of the cells to engraft and repopulate the bone marrow but did not increase the number of these cells during culturing. The hypothesis that the use of a culturing condition that stabilizes HIF-1α and antagonizes the AhR protein would promote the engraftment capacity of the LT-HSC population, was thus accepted.