Supplemental Data
Figure S1. Cell sorting strategy to purify murine HSC and MPP.
Representative gating of HSC and MPP. HSCs are defined as Lin (CD2, CD3, CD4,
CD8, Mac1, Gr1, Ter119, B220)-, CD48-, Sca1+, cKit+, CD150+. MPPs are defined
as Lin (CD2, CD3, CD4, CD8,Mac1,Gr1, Ter119, B220)-, CD48-, Sca1+, cKit+,
CD150-. 10,000 HSC and MPP were simultaneously sorted into 350 μl RLT buffer
(QIAGEN) directly for RNA isolation.
Figure S2. Ectopic expression of Hoxa5 in mouse fetal liver HSCs.
(A). Schematic diagram of transplantation strategy. (B). Transduction efficiency of
retro-viral infection of Hoxa5 and GFP control. Fetal liver HSCs (E14.5) were
enriched by AutoMACS. After two rounds of spin-infection, transduction rate were
detected by flow cytomerty. (C) Real time PCR confirmed the expression pattern of
Hoxa5 in GFP positive bone marrow cells of recipient mice. One million GFP
positive bone marrow cells of Hoxa5-GFP or GFP control were sorted. 2 μg RNA was
reversely transcripted into cDNA using oligo (T) universal primer. 25ng cDNA were
used as input of q-RT-PCR(Bio-Rad). Relative fold was calculated by value of
2^(-ΔΔCt). The value of GFP-Ctr was normalized to 1 for comparison purpose. Error
bar indicates standard Error. (**P<0.01). (D) Lineages analysis of Peripheral blood of
Secondary recipients of Hoxa5-HSCs.
Figure S3. Decreased pool of HSC/MPP, and reduced CFU counts of bone
marrow cells from recipient mice 6-month posttransplantation.
(A) Flow cytometry analysis of HSC and MPP in bone marrow cells isolated from
recipients transplanted with Hoxa5-HSC and GFP control. Plots from one
representative experiment were shown. (B) Statistic anlaysis of absolute MPPs and
HSCs from 100 million bone marrow nucleated cells. **, p<0.01. (C) Colony forming
assay indicates that bone marrow cells overexpressing Hoxa5 produce much less
colonies in vitro. 40,000 total bone marrow cells or sorted GFP+ bone marrow cells
from Hoxa5-GFP and control mice were planted in semisolid medium. The data
showed colony numbers counted at Day 8. Error bar means standard error. *, P<0.05;
**, P<0.01 (n=3).
Gene Symbol
Slamf1
Gdf3
Fhl1
Vwf
Tjp1
Cdkn1c
Apoe
Slc6a15
Socs2
Sdc2
Tgm2
Trpc6
Ndn
Jam3
Ppic
Grb10
Mllt3
Rab27b
Tek
Dach1
Cttn
Transcription per
Million
HSC
MPP
63.94
0.00
22.77
0.00
71.41
7.63
62.89
7.72
9.76
1.32
75.58
11.94
867.43
209.32
26.84
7.55
38.90
11.61
23.77
7.54
307.34
104.91
67.34
23.69
146.00
53.90
44.23
16.50
82.56
35.41
69.49
30.16
176.82
87.31
20.22
10.12
44.36
23.41
46.67
24.76
83.55
47.98
Table S1. RNA-seq results of well-known genes differentially expressed in HSC
vs MPP.