Evidence for SCID-Repopulating Cell Activity of CD34-Negative Cells by Intra-Bone Marrow

1Supplementary Information:

2Supplement Figure Legends:

3Figure S1. The limit of detection of human CD45+ cells in mouse BMs by FCM.

4In order to confirm the limit of detection of human CD45+ cells in mouse BMs by flow

5cytometry, we made test samples using mouse whole BM cells and human CB-derived

6mononuclear cells. The mixing ratios of murine to human cells were as follows: 0:100,

750:50, 90:10, 99.9:0.1, 99.99:0.01, 99.995:0.005 and 100:0. As clearly seen, we were

8able to detect 0.005% human CD45+ cells in the test samples. Based on these results, we

9scored the mice as positive if more than 0.01% of the total murine BM cells were

10human CD45+ cells.

12Figure S2. Screening of candidate positive markers for CB-derived CD34- SRCs

13(A) Candidate positive markers, including known HSC markers and various adhesion

14molecules, were screened by multicolor FCM using highly purified CB-derived 18Lin-

15CD34+/- cells. Known HSC markers, including Tie2, KDR, ABCG2, and CD150 were

16not expressed on these 18Lin-CD34+/- cells. On the other hand, CD133, c-kit, CD90,

17CXCR4, CD49f and CD93 were expressed on these 18Lin-CD34+/- cells. Back-gated

18scattergrams of the positive fractions of the above-mentioned markers, including

19CD133, c-kit, CD90, CXCR4, CD49f and CD93, in the 18Lin-CD34- cells were made

20into FSC/SSC dot grams. Each of the back-gated cells is shown in red (B). The blast-

21lymphocyte windows are shown by solid blue lines.

23Figure S3. The expression levels of lineage markers in the cocultures of 18Lin-

24CD34+/-CD133+/- cells with DP MSCs.

25The expression of CD33 (A), CD11b (B), CD14 (C), and CD41 (D) was analyzed by

26FCM. The percentages of positive cells are shown by solid columns. Each coculture

27contained six wells. The data represent the means ± SD. The statistical analysis was

28performed using two-tailed Student’s t-test. **P <0.01, n.s., not significant

30Figure S4. The secondary multi-lineage reconstitution abilities of CD34+CD133+

31and CD34- CD133+ SRCs.

32First, the R1 gate was set on the total murine BM cells obtained from these two

33representative NOG mice that received (A) CD34+CD133+ SRCs (upper column) and

34(B) CD34- CD133+ SRCs (lower column) 18 weeks after secondary transplantation.

35Then the living cells were gated as R2. Thereafter, the human CD45+ cells were gated as

36R3 (solid line). The expression of surface markers, including CD33 and CD19, on the

37R3-gated cells was analyzed by multicolor FCM. The percentages of positive cells in

38each scattergram are indicated.

40Figure S5. The expression patterns of CD93 and CD133 on Lin-CD45+CD34-

41CD38hi/lo/- cells and 18Lin-CD34-CD38 hi/lo/- cells.

42The expression patterns of CD93 and CD133 on Lin-CD45+CD34-CD38hi/lo/- cells and

4318Lin-CD34-CD38hi/lo/- cells were precisely analyzed by seven-color FCM. In this

44experiment, we used FITC-conjugated 18 Lin mAbs, Brilliant Violet 421-conjugated

45anti-CD34, PE-Cy7-conjugated anti-CD38, Brilliant Violet 510-conjugated anti-CD45,

46PE-conjugated anti-CD93, APC-conjugated anti-CD133 mAbs and 7-AAD (see Table

47S1). (A) The forward scatter/side scatter (FSC/SSC) profile of the immunomagnetically

48separated Lin- cells. The R1 gate was set on the blast-lymphocyte window. (B) The R2

49gate was set on the living cells. (C) The R3 gate was set on the human CD45+ cells. (D)

50The Lin-CD45+ cells were subdivided into three fractions: Lin-CD45+CD34+/-CD38hi

51(R4), Lin-CD45+CD34+/-CD38lo (R5), and Lin-CD45+CD34+/-CD38- (R6), according to

52their expression levels of CD38. (E) The Lin-CD34+/-CD38hi cells residing in the R4 gate

53were further subdivided into two fractions: Lin-CD38hiCD34-CD93+ (R7) and Lin-

54CD38hiCD34-CD93- (R8) cells. (F) The Lin-CD34+/-CD38lo cells residing in the R5 gate

55were further subdivided into two fractions: Lin-CD38loCD34-CD93+ (R9) and Lin-

56CD38loCD34-CD93- (R10) cells. (G) The Lin-CD34+/-CD38- cells residing in the R6 gate

57were further subdivided into two fractions: Lin-CD38-CD34-CD93+ (R11) and Lin-CD38-

58CD34-CD93- (R12) cells. (H) The Lin-CD38hi CD34-CD93+ (R7) cells were further

59subdivided into three fractions: 18Lin-CD38hi CD34-CD93+CD133+ (R13), 18Lin-

60CD38hiCD34-CD93+ CD133- (R14), and 18Lin+CD38hi CD34-CD93+ CD133- (R15) cells.

61(I) The Lin-CD38hiCD34-CD93- (R8) cells were further subdivided into three fractions:

6218Lin-CD38hi CD34-CD93- CD133+ (R16), 18Lin-CD38hi CD34-CD93-CD133- (R17),

63and 18Lin+CD38hiCD34-CD93- CD133- (R18) cells. (J) The Lin-CD38loCD34-CD93+

64(R9) cells were further subdivided into three fractions: 18Lin-CD38loCD34-

65CD93+CD133+ (R19), 18Lin-CD38loCD34-CD93+CD133- (R20), and

6618Lin+CD38loCD34-CD93+ CD133- (R21) cells. (K) The Lin-CD38loCD34-CD93- (R10)

67cells were further subdivided into three fractions: 18Lin-CD38loCD34-CD93-CD133+

68(R22), 18Lin-CD38loCD34-CD93-CD133-(R23), and 18Lin+CD38loCD34-CD93-CD133-

69(R24) cells. (L) The Lin-CD38-CD34-CD93+ (R11) cells were further subdivided into

70three fractions: 18Lin-CD38-CD34-CD93+CD133+ (R25), 18Lin-CD38-CD34-

71CD93+CD133- (R26), and 18Lin+CD38-CD34-CD93+ CD133- (R27) cells. (M) The Lin-

72CD38-CD34-CD93- (R12) cells were further subdivided into three fractions: 18Lin-

73CD38-CD34-CD93-CD133+ (R28), 18Lin-CD38-CD34-CD93-CD133-(R29) and

7418Lin+CD38-CD34-CD93-CD133- (R30) cells. The Lin-CD45+CD34-CD38-CD93+ (R11)

75cells (Bonnet’s study) did not express CD133, as they reported. However, almost all of

76these cells were included in the 18Lin+ cell fraction. On the other hand, 18Lin-CD34-

77CD38lo/-CD133+ (R22 and R28) cells (this study) were only detected in the CD93- cell

78fraction (R10 and R12). The percentages of the cells are indicated in each of the

79scattergrams.