Supplementary MaterialsSupplementary Figures 41598_2017_11909_MOESM1_ESM. Analysis of cultured cells after 2 days revealed that LSK cells lost NS-GFP intensity during differentiation (Fig.?1e), confirming the relationship between NS-GFP intensity and hematopoietic differentiation status. NS-GFP intensity is usually highest in LT-HSCs Next, we evaluated NS-GFP intensity among LSK cells as HSPCs. LSK cells can be subfractionated, based on their expression of SLAM family markers (i.e., CD150 and CD48), into LT-HSCs (HSC: CD150+CD48?LSK), MPP (CD150?CD48?LSK), and restricted progenitors (HPC1: CD150?CD48+LSK and HPC2: CD150+CD48+LSK). The LT-HSC population showed the highest NS-GFP intensity of these progenitor cell populations (Fig.?2a,b). Because another important indicator of LT-HSCs is usually CD34, we compared NS-GFP intensity between CD150+CD48? CD34?LSK cells and CD150+CD48? CD34+LSK cells. Although both populations showed Aplaviroc high levels of NS-GFP, the intensity of NS-GFP in CD150+CD48?CD34?LSK cells was higher than that in CD150+CD48?CD34+LSK cells (Fig.?2c). Thus, the level of NS-GFP expression corresponds with the expression of previously described HSC markers. Open in a separate window Physique 2 SLAM markers identify LT-HSCs that show the highest NS-GFP intensity. (a) Identification of HSCs using the CD150 and CD48 staining profile of Lin?Sca-1+c-Kit+ bone marrow cells. (b) The highest NS-GFP intensity was detected in the HSC population, with gradual decline in multipotent progenitors (MPP) and restricted progenitors (HPC1 and HPC2). (c) Among CD150+CD48? LSK cells, NS-GFP intensity is usually higher in CD34? than in CD34+ cells. Data shown are the average ratios??SD of median NS-GFP intensity of individual subpopulation, relative to HSCs in (b) and CD34? cells in (c), respectively (n?=?3). **(Fig.?5b), cells expressing less GFP (NS-GFP1+ and NS-GFP2+) did not have long-term reconstitution capacity (Fig.?5c), indicating that most of these cells are progenitors. Cells expressing higher levels of GFP (NS-GFP3+ and NS-GFP4+) showed greater repopulating capacity, but the frequency of NS-GFP4+-derived hematopoietic cells was much higher than that of NS-GFP3+-derived cells. Differentiation marker analysis showed that only NS-GFP4+ produced B cells, T cells, and Aplaviroc myeloid lineage cells (Fig.?5c), although the colony-forming abilities of NS-GFP4+ and NS-GFP3+ cells were comparable. Thus, the NS-GFP4+ subpopulation highly enriched cells with greater repopulating capacity, suggesting that NS-GFP expression can be used to purify LT-HSCs. Open in a separate window Physique 5 Repopulation capacity of the HSPC populations with different NS-GFP intensity. (a) FACS pattern of bone marrow LSK separation into four fractions according to NF-GFP intensity. (b) An colony formation assay shows no clear difference between the four LSK fractions. (c) After transplantation of the four fractions into lethally irradiated hosts (1,000 cells were transplanted per mouse), NS-GFP 4+ had the highest reconstitution capacity with multilineage differentiation potential. Data shown are the mean frequencies of Ly5.2+ cells in the peripheral blood and the mean frequencies of Ly5.2+ cells among B cells, T cells or myeloid cells??SD (n?=?3). **[liquid culture Progenitor cells (c-Kit+ Lineage?, 1??104) isolated from BM of NS-GFP tg mice Vamp5 were cultured for 2 days in RPMI 1640 made up of 20% FBS, 10 ng/ml recombinant murine (rm)IL-3, and 10 ng/ml rmIL-6 at 37?C in humidified air containing 5% CO2. Colony formation assay LSK fractions isolated from NS-GFP tg mice (2??103 cells each) were cultured for 7 days in semisolid medium containing 50 ng/ml recombinant murine stem cell factor (rmSCF), 10 ng/ml rmIL-3, 10 ng/ml rmIL-6, and 3 U/ml recombinant human erythropoietin (rhEPO) (Methocult GF M3434, Stem Cell Technologies) at 37?C in humidified air containing 5% CO2. Quantitative RT-PCR analysis RNA samples were purified from fractionated leukaemia cells (1??104) using an RNeasy kit (QIAGEN) and reverse-transcribed using an Advantage RT-for-PCR kit (Clontech, Takara Bio Inc.). PCR for NS was performed using a Dice Aplaviroc PCR Thermal Cycler (Takara Bio Inc.) as previously reported16. Statistics Unless otherwise stated, statistical differences between two groups were decided using unpaired Students between indicated groups are shown in each physique. Microarray study design Mouse LSK cells were sorted into four fractions: (a) NS-GFP1+ (lowest quartile of GFP intensity), (b) NS-GFP2+, (c) NS-GFP3+, and (d) Aplaviroc NS-GFP4+ (highest quartile). Total RNA from 1??104 cells in each fraction was isolated by using TRIzol (Invitrogen)..