Flow cytometric discrimination of mesenchymal progenitor cells from bone marrow-adherent cell populations using CD34/44/45(-) and Sca-1(+) markers.
BACKGROUND
Cultured bone marrow adherent cells (BMACs) have been commonly used as stem cells in bone and cartilage regeneration therapy. However, BMACs are actually a heterogeneous cell population, and clinicians might have previously transplanted more fibroblasts or other cells than actual stem cells. The purposes of this study were to (1) isolate immature mesenchymal stem cells with CD34/44/45 and Sca-1 surface-antigen patterns from BMACs using flow-activated cell sorting and (2) investigate their differentiation potential.


METHODS
Bone marrow cells were extracted from the mouse femur and cultured. Adherent cells could be identified approximately 3 days after seeding, and nonadherent cells were removed with the medium when it was changed. BMAC samples were cultured for 3, 7, 10, 14, 21, 28, 35, and 42 days after the first seeding. We directly isolated CD34/44/45(-)Sca-1(+) mesenchymal progenitor cells (MPC1) and CD34/45(-)/44(+) Sca-1(+) mesenchymal progenitor cells (MPC2) from BMACs based on their cell surface marker patterns using a fluorescence-activated cell sorter. These subgroups - MPC1, MPC2, and the residual cells in BMACs (non-MPC population: RCs) - were then induced to differentiate into bone, cartilage, and fat using a plate culture. The cultures were examined after histochemical staining on day 14.


RESULTS
In a plate culture, the MPC1 population had higher potential to differentiate into osteoblasts, chondrocytes, and lipocytes; whereas MPC2 and RCs differentiated into only two lineages: osteoblasts and lipocytes. The incidence of these multipotential cells was less than 5% among the cultured BMACs. MPC1 proliferated up to 17-fold within 3-4 weeks after separation from floating cells and did not increase thereafter.


CONCLUSIONS
BMACs are conventionally thought to differentiate into cartilage only in pellet culture, but we showed that MPC1 produced cartilage-like extracellular matrix in plate culture. MPC1, which are more immature cells than MPC2 and RCs, were multipotential progenitors that showed unique cartilage-differentiation potential. MPC1 had less ability to proliferate in BMAC culture, but they might have higher potential for chondrogenic differentiation.
Hachisuka
Hiroki
H
Department of Orthopedic Surgery, Programs for Applied Biomedicine, Division of Clinical Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Mochizuki
Yu
Y
Yasunaga
Yuji
Y
Natsu
Koji
K
Sharman
Patrick
P
Shinomiya
Rikuo
R
Ochi
Mitsuo
M
D000818:Animals; D018952:Antigens, CD34 / Q000502:physiology; D000950:Antigens, Ly / Q000502:physiology*; D002452:Cell Count; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D018960:Hyaluronan Receptors / Q000502:physiology; D017493:Leukocyte Common Antigens / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL
D018952:Antigens, CD34; D000950:Antigens, Ly; D018960:Hyaluronan Receptors; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D017493:Leukocyte Common Antigens