Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells

Abstract

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture.

© Mary Ann Liebert, Inc.

Figures

FIG. 1.

Rich vascularity in the anterior cruciate ligament (ACL) tissue. Arthroscopic findings show the representative ruptured site with hematoma (A) and tissue including septum divided by anteromedial and posterolateral bundles (B). Color images available online at www.liebertonline.com/scd

FIG. 2.

Vascular cells of fetal ACL. In fetal ACL tissue, there clearly exists the septum dividing the anterior medial (AM) and PL bundles (A) (×100). CD34 and CD146-positive cells (red) were located surrounding α-smooth muscle actin (α-SMA) (green)-positive arterioles in the septum region (B, C). In the midsubstance, CD34 and CD146-positive cells were found in the region without α-SMA-positive arterioles (B, C). Scale bar: 50 μm. Color images available online at www.liebertonline.com/scd

FIG. 3.

(A) H&E staining showed more vascular-like structures in the septum and ruptured site compared with the midsubstance. Arrow: vascular-like structure. Scale bar: 50 μm. (B) α-SMA staining recognized these structures as blood vessels. Scale bar: 50 μm. (C) The number of α-SMA-positive cells was significantly greater in the ruptured and septum regions than the midsubstance region. *P<0.05. (D) CD34-positive cells (red) located in α-SMA-positive arterioles (green) were abundantly found in the ruptured and septum regions compared with the midsubstance region. (E) CD146-positive cells (red) located surrounding α-SMA (green)-positive arterioles were also abundantly found in the ruptured and septum regions compared with the midsubstance region. (F, G) Fluorescence-activated cell sorting analysis demonstrated significantly higher numbers of CD34+ (F) and CD146+ (G) cells at the ruptured site compared with the midsubstance region. **P<0.01. The insets in figure A indicate the lesion shown in Figure B. Color images available online at www.liebertonline.com/scd

FIG. 4.

(A) ACL-derived cells from the site of ACL rupture and mid-substance region were sorted for expression of CD34 and CD146 after gating out hematopoietic (CD45-positive) cells. (B) CD34+CD45− cell fraction lost their CD34 expression and showed positive expression for CD105, CD44, CD90, and CD73 after 2 weeks of expansion. (C) All these cell populations showed positive expression for CD105, CD44, CD90, and CD73 and negative expression for CD45, CD133, CD56, and CD34. (D) After moderate expansion over 4 passages, CD34+CD45− cells exhibited significantly higher population doublings than the other groups in all passages. **P<0.05 for CD34+ versus CD146+ and CD34−CD146−, *P<0.05 for CD146+ versus CD34−CD146−.

FIG. 5.

(A) In monolayer culture, all population showed positive ALP staining; however, CD34+ cells revealed a larger number of ALP-positive cells than CD146+ cells and CD34−CD146− cells. Scale bar: 200 μm. (B) Mineralized nodular structures observed by von Kossa staining showed a greater number of positive cells in the CD34+ cell population than the other populations. Arrow: von Kossa positive. Scale bar: 200 μm. (C) Micro-CT analysis showed greater mineralization in the CD34+ cell population compared with the other populations. Scale bar: 500 μm. (D) Bone volume and density of CD34+ cells pellets were significantly larger than the other populations. **P<0.01, *P<0.05. (E) The mRNA expressions of collagen type IA2 (COL I) and osteocalcin were detected from pellets in all populations. (F) mRNA expression of COL I and osteocalcin was detected from pellets in all cell populations. The expression ratio of COL I to β-actin was significantly greater in the CD34+ and CD146+ cell populations than in the CD34−CD146− cell population. The expression ratio of osteocalcin to β-actin was significantly greater in the CD34+ cell population than in the CD34−CD146− cell population. **P<0.01, *P<0.05. Color images available online at www.liebertonline.com/scd

FIG. 6.

(A, B) Although all cell populations formed pellets, CD34−CD146− cells showed significantly larger sizes than the other populations. (C) Pellets of CD34−CD146− cells appeared to be stained well with Alcian blue compared with the other populations. (D) mRNA expression of COL II and aggrecan was detected from pellets in all populations. (E) There were no significant differences in the expression ratio of COL II to β-actin and aggrecan to β-actin among all cell populations. *P<0.05. Color images available online at www.liebertonline.com/scd

FIG. 7.

(A) Although all populations were positively stained for Oil Red O. (B) The number of Oil Red O-positive cells demonstrated that the CD34+ cells showed significantly more adipose cells (lipid droplets) compared with the other populations. Scale bar: 100 μm. (C) mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) was detected in all populations. (D) There were no significant differences in the expression ratio of PPARγ to β-actin among all cell populations. The expression ratio of LPL to β-actin was significantly greater in the CD34+ and CD146+ cell populations than in the CD34−CD146− cell population. **P<0.01, *P<0.05.

FIG. 8.

(A) All populations showed double-positive staining for acetylated low-density lipoprotein uptake and binding of Ulex europeus lectin. Scale bar: 100 μm. (B) All populations also showed vascular tube-like structures. Scale bar: 500 μm. (C) Assessment of tube length demonstrated that CD34+ cells showed a significantly higher potential for endothelial differentiation compared with the other populations. (D) mRNA expression of CD31 and VE-cadherin (VE-cad) was detected in all populations. (E) There were no significant differences in the expression ratio of VE-cad to β-actin among all cell populations. The expression ratio of CD31 to β-actin was significantly greater in the CD34+ and CD146+ cell populations than in the CD34−CD146− cell population. **P<0.01, *P<0.05. Color images available online at www.liebertonline.com/scd