Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells.
OBJECTIVE
We have successfully identified human cord blood (CB)-derived CD34-negative (CD34(-)) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphomyeloid repopulating ability using the intrabone marrow injection method. In our previous study, a limiting dilution analysis demonstrated the frequency of CD34(-) SRCs in CB-derived 13lineage-negative (Lin(-)) CD34(-) cells to be approximately 1/25,000. In this study, we intended to develop a high-resolution purification method to obtain highly purified CD34(-) SRCs.


METHODS
The pooled CB-derived Lin(-) cells were stained with 13 reported Lin monoclonal antibodies (mAbs) and 5 more Lin mAb, against CD11b, CD33, CD66c, CD45RA, and CD127. Then 18Lin(-)CD34(high), 18Lin(-)CD34(-), and 13Lin(-)CD34(high)CD38(-) cells were sorted by fluorescence-activated cell sorting. Stem cell characteristics of these three fractions of cells were analyzed by in vitro cultures and in vivo repopulation assays for evaluation of this new purification method.


RESULTS
A limiting dilution analysis demonstrated the frequency of CD34(-) SRCs in these 18Lin(-)CD34(-) cells to be approximately 1/1,000, which is associated with a seeding efficiency 25 times greater than the previous method. All primary recipient nonobese diabetic/Shi-scid/IL-2Rγc(null) mice that received transplants of only two CD34(-) SRCs were highly engrafted with human lymphomyeloid cells at 24 weeks after primary transplantation and showed secondary multilineage repopulating abilities.


CONCLUSIONS
We succeeded to highly purify the CD34(-) SRCs using 18Lin mAbs and the intrabone marrow injection technique. This newly developed high-resolution purification method is indispensable to precisely characterize a distinct class of primitive human CB-derived CD34(-) hematopoietic stem cells.
Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Moriguchi, Osaka, Japan.
Mari
M
Ishii
Yoshikazu
Y
Matsuoka
Yutaka
Y
Sasaki
Ryusuke
R
Nakatsuka
Masaya
M
Takahashi
Tsuyoshi
T
Nakamoto
Katsuhiko
K
Yasuda
Kazuo
K
Matsui
Hiroaki
H
Asano
Yasushi
Y
Uemura
Takashi
T
Tsuji
Shirou
S
Fukuhara
Yoshiaki
Y
Sonoda
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005260:Female; D005312:Fetal Blood / Q000737:chemistry / Q000166:cytology*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D016513:Mice, SCID; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016511:Severe Combined Immunodeficiency
D018952:Antigens, CD34