Adult stem cells were isolated and identified from hACLs and hMCLs. We showed that these hACL-SCs and hMCL-SCs exhibit characteristic stem cell properties, including clonogenicity, self-renewal, and multi-potency. Furthermore, both populations expressed stem cell markers nucleostemin, SSEA-4, STRO-1, and Oct-4 as well as several CD markers (CD44 and CD90) for mesenchymal stem cells (MSCs), but not those for endothelial cells, hematopoietic stem cells, leukocytes, or pericytes (CD31, CD34, CD45, and CD146). However, it was found that a smaller proportion of hACL-SCs expressed STRO-1, Oct-4, and CD44 compared to hMCL-SCs. hACL-SCs also grew about 50% slower and formed smaller and fewer colonies than hMCL-SCs. Moreover, there was a marked difference in long-term self-renewal capability between the two types of stem cells: hACL-SCs became differentiated after only five passages and two months in culture, whereas hMCL-SCs maintained a nearly undifferentiated state even after 13 passages and the same culture time. Taken together, these results show that hACL-SCs and hMCL-SCs are ligament specific stem cells that possess intrinsically different stem cell properties.
Nucleostemin, SSEA-4, STRO-1, and Oct-4 are four well established stem cell markers used to confirm the stem cell identity of hACL-SCs and hMCL-SCs in this study. Nucleostemin is a nucleolar protein believed to act via p53 [25, 26] and to be expressed by stem cells and cancer cells but not terminally differentiated cells [26, 27]. Thus, high levels of nucleostemin expression by hACL-SCs and hMCL-SCs in this study were indicative of proliferating, self-renewing populations of ASCs. Like nucleostemin, Oct-4 is also a nuclear protein expressed in embryonic stem cells and carcinoma cell lines but not in differentiated cells . Oct-4 expression is lost during the process of differentiation, and downregulation of Oct-4 is thought to directly induce stem cell differentiation [29–31].
SSEA-4 is a member of the stage specific antigen family first identified as a marker that disappeared from human teratocarcinoma cells as they differentiated [32, 33], which has since been recognized as a marker of human embryonic stem cells  and mesenchymal stem cells  as well. STRO-1 is a cell surface antigen found on bone marrow mononuclear cells  capable of differentiating down osteogenic [37, 38], chondrogenic, and adipogenic lines . In addition, STRO-1 is expressed in human periodontal ligament stem cells .
In addition to the stem cell markers above, we examined the expression of CD surface markers on hACL-SCs and hMCL-SCs. Both ACL-SCs and MCL-SCs expressed CD44 and CD90 (albeit the former exhibited a less extent than the latter). Neither of the two types of ligament stem cells expressed CD31, CD34, CD45, or CD146. CD44 is a common MSC antigen [41, 42] and is used as a marker for bone marrow stem cells (BMSCs) . CD90 is a fibroblast marker that has also been found on undifferentiated human embryonic stem cells , and human MSCs are consistently positive for both CD44 and CD90 [44–46]. Endothelial cell marker CD31 , hematopoietic stem cell marker CD34 , pericyte marker CD146 , and leukocyte marker CD45  were expressed by neither hACL-SCs nor hMCL-SCs. These results provide additional evidence that hACL-SCs and hMCL-SCs are ASCs of mesenchymal origin.
A recent study by Cheng et al. looked into the possibility of populations of stem cells existing in human ACLs . It was shown that cells isolated from the ACL are clonogenic with multidifferentiation potential and express surface markers similar to MSCs, including CD73, CD90, and CD105. The hACL-SCs isolated in our study displayed similar characteristics as the ligament stem cells from Cheng et al.'s study in terms of clonogenicity, multipotency, and expression of stem cell markers CD44 and CD90, but not CD34 or CD45. However, unlike this study, the ACL samples used by Cheng et al. may not be normal as they were collected from patients who had undergone total knee arthroplasty.
Another study, in which ligament cells were derived from young rabbits, found that the chondrogenic potential of 'ligament-derived cells' was greater for ACL cells than MCL cells . However, their study used a mixed cell population rather than isolated stem cells as this study did. Finally, tissue specific stem cells, such as hACL-SCs and hMCL-SCs, have been found in various tissues, including bone marrow , the periodontal ligament , and human, mouse, and rabbit patellar tendons [22, 23], which are similar to extra-articular ligaments. In addition, rat flexor tendons were found to contain stem cells . Our group has also shown that stem cells from rabbit patellar and Achilles tendons express nucleostemin, Oct-4, and SSEA-4 . The same stem cell markers were found to be expressed on both hACL-SCs and hMCL-SCs in this study.
While both hACL-SCs and hMCL-SCs were shown to be ASCs, differences were also observed with regard to their clonogenicity, self-renewal capacity, and differentiation potential. We previously also found that stem cells derived from rabbit patellar and Achilles tendons exhibit marked differences in colony formation and cell proliferation rate . Differences in gene profiles have also been noted between MSCs derived from human intra-articular (synovium, meniscus, and ACL) and extra-articular sources (adipose, muscle, and bone) . In addition, previous studies showed that ACL fibroblasts proliferate more slowly than their MCL counterparts [9, 53]. These above studies, however, differ from this study in that they used mixed cultures; in other words, the cell population could include both stem cells and residential adult cells (for example, ACL fibroblasts). Nevertheless, the finding in this study that hACL-SCs proliferate much more slowly than hMCL-SCs (Figure 2) is largely consistent with conclusions of the above studies.
It is well recognized that injured ACLs have a low healing capacity, whereas injured MCLs display a high healing capacity [54–56]. Given that ASCs are the body's natural reservoir for replenishing pools of specialized cells that have been damaged in tissue injury, we suggest that the differential characteristics of hACL-SCs and hMCL-SCs found in this study may also contribute to their respective ligaments' differential healing capacities. Specifically, our data seem to indicate that hACL-SCs lose their 'stemness' earlier than hMCL-SCs. This may contribute to non-healing of injured ACLs as the hACL-SCs may have lost their ability to self-renew during the healing process; as a result, few cells will be available for repair of the injured ACLs. On the other hand, because of their superior capability of self-renewal, hMCL-SCs can continuously supply cells to effectively repair injured MCLs.
Besides the inherent lower stem cell capacity of hACL-SCs, blood flow, an 'external' factor, is known to be lower in the ACL than in the MCL in both intact and injured states. As a result, fewer nutrients will be available to hACL-SCs compared to hMCL-SCs. Therefore, again a smaller number of hACL-SCs and their progeny cells will be produced compared to hMCL-SCs.
The finding that the hACL contains ASCs may enable one to devise a new tissue engineering approach for repair of injured hACLs. This could be done by using small portions of ligaments to isolate and expand hACL-SCs in vitro and then implanting the cells into the injured ACL. On the other hand, while the injured MCL heals spontaneously, the quality of the healed tissue is still inferior with scar formation . This is true even with implantation of natural scaffolding materials [58, 59]. Therefore, hMCL-SCs may also be used as a source for cellular-based therapies to restore the structure and function of the injured MCL.
Several comments are now in place regarding the proper interpretation of the results of this study. First, we used local application of trypsin to isolate stem cell colonies in cultures. Such a technique may be subject to contamination of a small number of ligament fibroblasts; in other words, the stem cell populations used in this study may not be pure. Second, there is an apparent difference in the results of stem cell marker expression between immunocytochemistry (Figure 3) and FACS analysis (Table 3). The difference may be caused by the different passages of cells and the number of donors used in the two different methods. For immunostaining we used hACL-SCs and hMCL-SCs at passage 1 from a 26 year old donor, but for FACS analysis, passages 2 to 3 from six donors were used and the results were represented by mean ± SD. Third, hACL-SCs and hMCL-SCs were found to express low levels of non-tenocyte related genes, including PPARγ, LPL, Sox-9, collagen II, and Runx-2, even without differentiation induction media. There are two possible reasons for this. While the ligaments used in our experiments were limited at grade 0 (normal), the donors might have slight degenerative changes in their ACL and MCL ligaments. Additionally, there is a possibility that a small population of stem cells that somehow had differentiated towards non-fibroblasts was present in cultures.