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

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. © 2012 Mary Ann Liebert, Inc

Immunofluorescence and FACS analysis reveals cells that co-express stromal and endothelial markers.

<p><b>A–A’’</b>. Wholemount immunofluorescence reveals that PECAM positive vessels (A, red) are present within the Foxd1 positive renal stroma (A’, green) on the merged image (A’’). <b>B.</b> IF in the cortex of an E18.5 kidney shows that developing endothelial cells stained with PECAM (blue) are present in the stroma stained with Foxd1 (green) but not in nephron precursors stained with Six2 (red). <b>B’–B’’’.</b> Higher power views from “B” reveal expression of the stromal marker Foxd1 in the nucleus (green, arrowhead) and PECAM in the cytoplasm (blue) of the same cell. <b>C–C’’.</b> High power image of IF at E18.5 shows co-expression of the stromal marker Foxd1 (green) and the endothelial marker Erg (red) in nuclei of some renal cells (arrowhead). <b>D.</b> Flow sorted kidneys from E18.5 <i>Foxd1EGFPcre</i> mouse shows co-expression of GFP (Foxd1-positive stromal marker, X axis) and Flk1 (Y axis) in a subset of cells (P8). Scale bars A = 50 µm, B and C = 10 µm.</p

Stromal derivatives form endothelium that is present in peritubular capillaries.

<p><b>A.</b> IF in E18.5 <i>Foxd1cre</i>CAG mouse section reveals RFP stromal derivatives (red) that co-label with Erg (green) in a peritubular capillary (arrowhead); in the glomerulus (G), the stromal-derived cells form the mesangium (red) but do not express Erg (green). <b>B.</b> Flow sorted kidneys from E18.5 <i>Foxd1cre</i>CAG reporter mice show co-expression of RFP (Foxd1-stromal derivative, X axis) and Flk1 (Y axis) in a subset of cells (P5). <b>C-C’’.</b> IF in a P30 <i>Foxd1cre</i>CAG kidney section reveals a peripheral peritubular capillary with co-expression of RFP stromal derivatives (red) and PECAM (green) (arrowhead). <b>D.</b> IF from a P30 <i>Foxd1cre</i>CAG kidney section shows a glomerulus (G) and major vessel (V), that express RFP stromal derivatives in the mesangium and vascular smooth muscle (red), respectively, but that do not co-express RFP and PECAM (green) in endothelium in those tissues. <b>E.</b> Flow sorted kidneys from P30 <i>Foxd1cre</i>CAG reporter mice show that many RFP stromal derived cells (X axis) co-express PECAM (Y axis) as shown in P5. Scale bars A = 30 µm, C = 20 µm, D = 50 µm.</p

Foxd1 positive lung cells differentiate into endothelial-like tubules and take up Ac-LDL.

<p><b>A–C.</b> Tubulogenesis assay: <b>A</b>. <i>Foxd1EGFPcre</i>/GFP positive sorted cells are rounded when cultured at day zero. <b>B.</b> After 7 days of growth, the PECAM positive populations remain rounded having lost the ability to form endothelial-like tubules. <b>C.</b> After 7 days of growth, <i>Foxd1EGFPcre</i>/GFP positive cells readily formed endothelial-like tubular networks. <b>D.</b> Acetylated LDL assay: Under hypoxic conditions, <i>Foxd1EGFPcre</i>/GFP cells have endocytosed Ac-LDL (green) (blue = DAPI). Scale bars A–C = 50 µm, D = 5 µm.</p