<p> 1</p><p>1Supplementary Information:</p><p>2Supplement Figure Legends:</p><p>3Figure S1. The limit of detection of human CD45+ cells in mouse BMs by FCM.</p><p>4In order to confirm the limit of detection of human CD45+ cells in mouse BMs by flow</p><p>5cytometry, we made test samples using mouse whole BM cells and human CB-derived</p><p>6mononuclear cells. The mixing ratios of murine to human cells were as follows: 0:100,</p><p>750:50, 90:10, 99.9:0.1, 99.99:0.01, 99.995:0.005 and 100:0. As clearly seen, we were</p><p>8able to detect 0.005% human CD45+ cells in the test samples. Based on these results, we</p><p>9scored the mice as positive if more than 0.01% of the total murine BM cells were</p><p>10human CD45+ cells. </p><p>11</p><p>12Figure S2. Screening of candidate positive markers for CB-derived CD34- SRCs</p><p>13(A) Candidate positive markers, including known HSC markers and various adhesion</p><p>14molecules, were screened by multicolor FCM using highly purified CB-derived 18Lin-</p><p>15CD34+/- cells. Known HSC markers, including Tie2, KDR, ABCG2, and CD150 were</p><p>16not expressed on these 18Lin-CD34+/- cells. On the other hand, CD133, c-kit, CD90,</p><p>17CXCR4, CD49f and CD93 were expressed on these 18Lin-CD34+/- cells. Back-gated</p><p>18scattergrams of the positive fractions of the above-mentioned markers, including</p><p>19CD133, c-kit, CD90, CXCR4, CD49f and CD93, in the 18Lin-CD34- cells were made</p><p>20into FSC/SSC dot grams. Each of the back-gated cells is shown in red (B). The blast-</p><p>21lymphocyte windows are shown by solid blue lines.</p><p>22</p><p>23Figure S3. The expression levels of lineage markers in the cocultures of 18Lin-</p><p>24CD34+/-CD133+/- cells with DP MSCs.</p><p>2 1 3</p><p>25The expression of CD33 (A), CD11b (B), CD14 (C), and CD41 (D) was analyzed by</p><p>26FCM. The percentages of positive cells are shown by solid columns. Each coculture</p><p>27contained six wells. The data represent the means ± SD. The statistical analysis was</p><p>28performed using two-tailed Student’s t-test. **P <0.01, n.s., not significant</p><p>29 </p><p>30Figure S4. The secondary multi-lineage reconstitution abilities of CD34+CD133+</p><p>31and CD34- CD133+ SRCs.</p><p>32First, the R1 gate was set on the total murine BM cells obtained from these two</p><p>33representative NOG mice that received (A) CD34+CD133+ SRCs (upper column) and</p><p>34(B) CD34- CD133+ SRCs (lower column) 18 weeks after secondary transplantation.</p><p>35Then the living cells were gated as R2. Thereafter, the human CD45+ cells were gated as</p><p>36R3 (solid line). The expression of surface markers, including CD33 and CD19, on the</p><p>37R3-gated cells was analyzed by multicolor FCM. The percentages of positive cells in</p><p>38each scattergram are indicated.</p><p>39</p><p>40Figure S5. The expression patterns of CD93 and CD133 on Lin-CD45+CD34-</p><p>41CD38hi/lo/- cells and 18Lin-CD34-CD38 hi/lo/- cells. </p><p>42The expression patterns of CD93 and CD133 on Lin-CD45+CD34-CD38hi/lo/- cells and</p><p>4318Lin-CD34-CD38hi/lo/- cells were precisely analyzed by seven-color FCM. In this</p><p>44experiment, we used FITC-conjugated 18 Lin mAbs, Brilliant Violet 421-conjugated</p><p>45anti-CD34, PE-Cy7-conjugated anti-CD38, Brilliant Violet 510-conjugated anti-CD45,</p><p>46PE-conjugated anti-CD93, APC-conjugated anti-CD133 mAbs and 7-AAD (see Table</p><p>47S1). (A) The forward scatter/side scatter (FSC/SSC) profile of the immunomagnetically</p><p>48separated Lin- cells. The R1 gate was set on the blast-lymphocyte window. (B) The R2</p><p>49gate was set on the living cells. (C) The R3 gate was set on the human CD45+ cells. (D)</p><p>4 2 5</p><p>50The Lin-CD45+ cells were subdivided into three fractions: Lin-CD45+CD34+/-CD38hi</p><p>51(R4), Lin-CD45+CD34+/-CD38lo (R5), and Lin-CD45+CD34+/-CD38- (R6), according to</p><p>52their expression levels of CD38. (E) The Lin-CD34+/-CD38hi cells residing in the R4 gate</p><p>53were further subdivided into two fractions: Lin-CD38hiCD34-CD93+ (R7) and Lin-</p><p>54CD38hiCD34-CD93- (R8) cells. (F) The Lin-CD34+/-CD38lo cells residing in the R5 gate</p><p>55were further subdivided into two fractions: Lin-CD38loCD34-CD93+ (R9) and Lin-</p><p>56CD38loCD34-CD93- (R10) cells. (G) The Lin-CD34+/-CD38- cells residing in the R6 gate</p><p>57were further subdivided into two fractions: Lin-CD38-CD34-CD93+ (R11) and Lin-CD38-</p><p>58CD34-CD93- (R12) cells. (H) The Lin-CD38hi CD34-CD93+ (R7) cells were further</p><p>59subdivided into three fractions: 18Lin-CD38hi CD34-CD93+CD133+ (R13), 18Lin-</p><p>60CD38hiCD34-CD93+ CD133- (R14), and 18Lin+CD38hi CD34-CD93+ CD133- (R15) cells.</p><p>61(I) The Lin-CD38hiCD34-CD93- (R8) cells were further subdivided into three fractions:</p><p>6218Lin-CD38hi CD34-CD93- CD133+ (R16), 18Lin-CD38hi CD34-CD93-CD133- (R17),</p><p>63and 18Lin+CD38hiCD34-CD93- CD133- (R18) cells. (J) The Lin-CD38loCD34-CD93+</p><p>64(R9) cells were further subdivided into three fractions: 18Lin-CD38loCD34-</p><p>65CD93+CD133+ (R19), 18Lin-CD38loCD34-CD93+CD133- (R20), and</p><p>6618Lin+CD38loCD34-CD93+ CD133- (R21) cells. (K) The Lin-CD38loCD34-CD93- (R10)</p><p>67cells were further subdivided into three fractions: 18Lin-CD38loCD34-CD93-CD133+</p><p>68(R22), 18Lin-CD38loCD34-CD93-CD133-(R23), and 18Lin+CD38loCD34-CD93-CD133-</p><p>69(R24) cells. (L) The Lin-CD38-CD34-CD93+ (R11) cells were further subdivided into</p><p>70three fractions: 18Lin-CD38-CD34-CD93+CD133+ (R25), 18Lin-CD38-CD34-</p><p>71CD93+CD133- (R26), and 18Lin+CD38-CD34-CD93+ CD133- (R27) cells. (M) The Lin-</p><p>72CD38-CD34-CD93- (R12) cells were further subdivided into three fractions: 18Lin-</p><p>73CD38-CD34-CD93-CD133+ (R28), 18Lin-CD38-CD34-CD93-CD133-(R29) and</p><p>7418Lin+CD38-CD34-CD93-CD133- (R30) cells. The Lin-CD45+CD34-CD38-CD93+ (R11)</p><p>6 3 7</p><p>75cells (Bonnet’s study) did not express CD133, as they reported. However, almost all of</p><p>76these cells were included in the 18Lin+ cell fraction. On the other hand, 18Lin-CD34-</p><p>77CD38lo/-CD133+ (R22 and R28) cells (this study) were only detected in the CD93- cell</p><p>78fraction (R10 and R12). The percentages of the cells are indicated in each of the</p><p>79scattergrams.</p><p>8 4</p>