Isolation of therapeutically functional mouse bone marrow mesenchymal stem cells within 3 h by an effective single-step plastic-adherent method.
OBJECTIVE
Isolation of mouse mesenchymal stem cells (mMSCs), by the approach of plastic adherence, has been difficult due to persistent contamination by haematopoietic cells (HCs); we have observed that this contamination was due to engagement between HCs and mMSCs. The HCs can be lifted together with the mMSCs despite their insensitivity to trypsin digestion. Herein, we provide a single-step procedure to rapidly segregate mMSCs from HC contaminants using transient lower-density plastic adherence (tLDA).


METHODS
The tLDA was performed by replating bone marrow adherent cells at lower density (1.25 x 10(4) cells/cm(2)) than usual, allowing for transient adherence of no more than 3 h, followed by trypsin digestion. tLDA-isolated cells were evaluated by immunophenotyping, multi-differentiation potentials, immunosuppressive properties, and therapeutic potential as demonstrated by symptoms of osteoporosis.


RESULTS
The single-step tLDA method can effectively eliminate the persistent HC contaminants; tLDA-isolated cells were phenotypically equivalent to those reported as mMSCs. The isolated cells possessed classic tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages and had immunosuppressive properties. After intravenous transplantation, they migrated into the allogeneic bone marrow and rescued hosts from osteoporosis symptoms, demonstrating their therapeutic potential.


CONCLUSIONS
We have developed a simple and economical method that effectively isolates HC-free, therapeutically functional mMSCs from bone marrow cell adherent cultures. These cells are suitable for various mechanistic and therapeutic studies in the mouse model.
Hsiao
F S-H
FS
Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan.
Cheng
C-C
CC
Peng
S-Y
SY
Huang
H-Y
HY
Lian
W-S
WS
Jan
M-L
ML
Fang
Y-T
YT
Cheng
E C-H
EC
Lee
K-H
KH
Cheng
W T-K
WT
Lin
S-P
SP
Wu
S-C
SC
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis / Q000378:metabolism; D016026:Bone Marrow Transplantation / Q000379:methods*; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D019070:Cell Lineage / Q000502:physiology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D016130:Immunophenotyping; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D010024:Osteoporosis / Q000628:therapy; D010969:Plastics / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D000954:Antigens, Surface; D010969:Plastics; D014357:Trypsin