Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity

Human ACL and MCL tissue samples free of pathology were obtained from six adult donors ranging in age from 20 to 36 years old (Table 1). The protocol for obtaining the ligament tissue samples was approved by the University of Pittsburgh Institutional Review Board. To prepare the tissue cultures, the ligament sheath was removed to obtain the core portion of the ligament, which was then minced into small pieces, and each 100 mg of wet tissue samples were digested in 1 ml of PBS containing 3 mg of collagenase type I and 4 mg of dispase as described previously [23]. Single cell suspensions were cultured in either a 96 well plate (1 cell/well) or T25 flasks (4 × 10⁵/flask). After eight to ten days in culture, hACL-SCs and hMCL-SCs formed distinct colonies on the plastic surfaces of the plates or flasks. The colonies were visualized with methyl violet and then counted with a hemocytometer.

Trypsin was locally applied to each colony under microscopic visualization in order to detach stem cell colonies, and detached cells were collected and transferred to T25 flasks for further culture. The growth medium consists of Dulbecco's modified Eagle's medium (DMEM; Lonza, Walkersville, MD) supplemented with 20% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 100 μM 2-mercaptoethanol (Sigma-Aldrich, St Louis), 100 U/ml penicillin, and 100 μg/ml streptomycin (Atlanta Biologicals, Lawrenceville, GA). To measure the proliferative capacities of hACL-SCs and hMCL-SCs, we used population doubling time (PDT) as an index. Briefly, hACL-SCs or hMCL-SCs were seeded in 6-well plates at a density of 6 × 10⁴/well and cultured with growth medium until confluence. The PDT is calculated by dividing the total culture time by the number of generations [23].

Immunocytochemistry was used to assay for expression of the following stem cell markers: nucleostemin, Oct-4, STRO-1, and SSEA-4. To perform immunostaining, hACL-SCs or hMCL-SCs were seeded in 12-well plates at a density of 3.5 × 10⁴/well and cultured with growth medium for 3 days. The medium was then removed, and the cells were washed with PBS once. The stem cells were first fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes. For nucleostemin and Oct-4 staining, this step was followed by washing with 0.1% Triton-X100 for 15 minutes. All cells were then blocked with 3% mouse serum for 1 hour. In the first antibody reaction, the stem cells were incubated with either mouse anti-human STRO-1 (1:400, Cat. #39-8401, Invitrogen, Carlsbas, CA), mouse anti-human SSEA-4 (1:400, Cat. #414000, Invitrogen, Carlsbas, CA), goat anti-human nucleostemin (1:350, Cat. # GT15050, Neuromics, Edina, MN), or rabbit anti-human Oct-3/4 (1:350, Cat. # sc-9081, Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 2 hours. After washing the cells with PBS, cyanine 3 (Cy3)-conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibody (1:500, Cat.# A10521, Invitrogen, Catlsbas, CA) was applied at room temperature for 1 hour to STRO-1 and SSEA-4 samples while Cy3-conjugated donkey anti-goat IgG secondary antibody (1:500, Cat.# AP180C, Millipore, Temecula, CA) was used for nucleostemin and Cy-3 conjugated goat anti-rabbit IgG antibody (1:400, Cat. # AP132C, Millipore, Temecula, CA) was used for Oct-3/4 samples at room temperature for 2 hours. The cells were also counterstained with Hoechst 33342 (Cat. # 33270; Sigma, St Louis).

In addition, stem cell surface markers CD31, CD44, CD90, CD34, CD45, and CD146 were stained in parallel by immunocytochemistry. Fixed cells were incubated with fluorescein isothiocyanate (FITC)- or Cy3- or phycoerythrin (PE)-conjugated mouse anti-human antibodies (1:400) for 1 hour. All steps were performed at room temperature. Unless otherwise noted, all antibodies were purchased from Chemicon International (Temecula, CA), BD Pharmingen (BD Biosciences; http://​bdbiosciences.​com), or Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescent images of the stained cells were taken by a CCD camera on an inverted fluorescent microscope (Nikon eclipse, TE2000-U) using SPOT™ imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI).

The multi-differentiation potentials of hACL-SCs and hMCL-SCs were examined in vitro to determine whether they could undergo adipogenesis, chondrogenesis, and osteogenesis. Cells at passage 1 were seeded in a 6-well plate at a density of 2.4 × 10⁵ cells/well in basic growth medium consisting of low glucose DMEM, 10% heat inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. After reaching confluence, cells for adipogenesis were cultured in adipogenic induction medium (Millipore, Cat. # SCR020) consisting of basic growth medium supplemented with 1 μM dexamethasone, 10 μg/ml insulin, 100 μM indomethacin, and 0.5 mM isobutylmethylxanthine (IBMX) for 21 days. Oil Red O assay was used to detect lipid droplets contained in the differentiated adipocytes.

For chondrogenesis, confluent stem cells were cultured in chondrogenic induction medium consisting of basic growth medium along with 40 μg/ml proline, 39 ng/ml dexamethasone, 10 ng/ml transforming growth factor beta 3 (TGF-β3), 50 μg/ml ascorbate 2-phosphate, 100 μg/ml sodium pyruvate, and 50 mg/ml ITS premix (BD, Cat. # 354350). After 21 days in culture, the glycosaminoglycans (GAG)-rich matrix produced by differentiated chondrocytes was stained using the Safranin O assay.

Finally, for osteogenic differentiation, stem cells were cultured in osteogenic induction medium consisting of basic growth medium with 0.1 μM dexamethasone, 0.2 mM ascorbic 2-phosphate, and 10 mM glycerol 2-phosphate for 21 days. The differentiated cells released calcium-rich deposits, which were stained by the Alizarin Red S assay. Cells cultured in basic growth medium for the same durations were used as a control.

After discarding the medium, the cells were washed 3 times for 5 minutes each with PBS. The cells were then fixed in 4% paraformaldehyde for 40 minutes at room temperature. Subsequently, the cells were washed 3 times with PBS at 5 minute intervals and then with water twice for 5 minutes each. Finally, the cells were incubated with 0.36% Oil Red O solution (Millipore, Cat. # 90358) for 50 minutes, followed by washing 3 times with water.

The cells were fixed in ice-cold ethanol for 1 hour, rinsed with distilled water twice for 5 minutes each, and stained with Safranin O solution (Sigma, Cat. # HT904) for 30 minutes. The cells were then rinsed five times with distilled water.

The cells cultured in osteogenic medium were fixed in chilled 70% ethanol for 1 hour, rinsed with distilled water for 5 minutes twice, and stained with Alizarin Red S solution (Millipore, Cat. # 2003999) for 30 minutes at room temperature. The cells then underwent five rinses with distilled water. Cells stained with the three assays were examined, and images were taken and analyzed on an inverted fluorescent microscope as noted earlier.

Briefly, 12 views from each well were randomly chosen on a microscope with a magnification of 20×. Then, the areas of positive staining were identified manually and computed by SPOT IMAGING Software. Next, the proportion of positive staining was calculated by dividing the stained area by the view area. Twelve ratio values for each of three wells were averaged to obtain the percentage of positive staining, which represents the extent of cell differentiation in the respective induction medium.

The cell suspension (2.5 × 10⁶ in 50 μl PBS) was incubated with 20 μl of the appropriate serum in a round-bottomed tube at 4°C for 30 minutes. Subsequently, 2 μl of primary antibody (0.2 mg/ml stock solution) was added and incubated at 4°C for 1 hour. The cells were then washed three times with 2% FBS-PBS and reacted with 1 μl of secondary antibody (1 mg/ml stock solution) at 4°C for 1 hour. Afterwards the cells were washed twice with PBS and then fixed with 0.5 ml of 1% paraformaldehyde. FACS analysis was performed with BD LSR II Flow Cytometer (BD Biosciences, http://​www.​bdbiosciences.​com).

RNA was extracted from hACL-SCs and hMCL-SCs using the RNeasy Mini Kit with an on-column DNase I digest (Qiagen). First-strand cDNA was synthesized from 1 μg total RNA, which was synthesized in a 20 μl reaction by reverse transcription using SuperScript II (Invitrogen). The following conditions for cDNA synthesis were applied: 65°C for 5 minutes and cooling for 1 minute at 4°C, then 42°C for 50 minute and 72°C for 15 minute. Next, qRT-PCR was performed using QIAGEN QuantiTect SYBR Green PCR Kit (QIAGEN). In a 50 μl PCR reaction mixture, 2 μl cDNA (total 100 ng RNA) were amplified in a Chromo 4 Detector (MJ Research, Maltham, MA) with incubation at 94°C for 5 minutes, followed by 30 to 40 cycles of a three temperature program of 1 minute at 94°C, 30 seconds at 57°C, and 30 seconds at 72°C. The PCR reaction was terminated after a 10 minute extension at 70°C and stored at 4°C until analysis. The following human-specific primers based on previous publications were used: Oct-4, STRO-1, peroxisome proliferator-activated receptor gamma (PPARγ, lipoprotein lipase (LPL), Sox-9, collagen type II (Coll. II), Runx2, and alkaline phosphatase (ALP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control (Table 2). All primers were synthesized by Invitrogen. The products (each 5 μl) from qRT-PCR were run on a 2% agarose gel in 0.5×TBE buffer at 100 V. The separated DNA fragments were analyzed by a gel documentation system (Bio-Rad).

For each experimental condition, at least three replicates were performed. The results presented in the figures are representative of these (mean ± SD, n = 3 to 6). Two-tailed student t-test was used for statistical analysis. A P-value less than 0.05 was considered to be significantly different.

After three days in culture, cells from single cell suspensions of both hACL and hMCL tissue samples attached to plate surfaces and formed colonies. However, the number and size of cell colonies from hACL-SCs and hMCL-SCs were markedly different: colonies formed by hACL-SCs were fewer in number (Figure 1A, B) and smaller in size than those of hMCL-SCs (Figure 1C, D). Moreover, hACL-SCs grew much more slowly than hMCL-SCs, as the PDT for hACL-SCs was nearly double that of hMCL-SCs (Figure 2).

Using immunocytochemistry, both hACL-SCs and hMCL-SCs were found to express nucleostemin (Figure 3A, B), SSEA-4 (Figure 3D, E), CD44 (Figure 3G, H), and CD90 (Figure 3J, K). There was no significant difference in nucleostemin expression between hACL-SCs and hMCL-SCs, and more than 95% of both stem cells stained positively for nucleostemin (Figure 3C). However, only 40% of hACL-SCs were positively stained by SSEA-4, whereas more than 56% of hMCL-SCs were positive stained (Figure 3F). Similarly, about 42% of hACL-SCs expressed CD44 compared to about 60% for hMCL-SCs (Figure 3I). Furthermore, both hACL-SCs and hMCL-SCs expressed high levels of CD 90 (Figure 3L). Immunostaining for CD31, CD34, CD45, and CD146 was negative (data not shown).

Moreover, hACL-SCs stained weakly for STRO-1, whereas more than 95% of hMCL-SCs stained positive for STRO-1 (Figure 4A). The gene expression level of STRO-1 in hACL-SCs was much lower than in hMCL-SCs (Figure 4B). Similarly, fewer than 40% of hACL-SCs expressed Oct-4, but more than 90% of hMCL-SCs stained positive for Oct-4 (Figure 4C). Finally, hACL-SCs expressed much lower levels of the Oct-4 gene than hMCL-SCs (Figure 4D).

In addition, FACS analysis results showed that the percentages of CD31, CD34, CD45, and CD146 positive cells were less than 2%. Moreover, while CD44, CD90, and SSEA-4 were expressed to a greater extent by both hACL-SCs and hMCL-SCs (Figure 5), there was a significant difference in the level of expression between the two types of stem cells (Table 3).

Both hACL-SCs and hMCL-SCs were able to undergo self-renewal, indicated by the maintenance of a cobblestone shape after repetitive passage and expression of stem cell markers nucleostemin and SSEA-4.

However, after five passages and two months in culture, hACL-SCs became elongated (Figure 6A), a typical fibroblast phenotype, and lost expression of nucleostemin and SSEA-4 (Figure 6C, E), indicating that they had undergone differentiation. In contrast, hMCL-SCs, even after 13 passages and two months of culture time, maintained a cobblestone shape (Figure 6B) and expressed a high level of nucleostemin and SSEA-4 (Figure 6D, F). The extent of nucleostemin expression at this passage, however, was lower than that at passage 1 (Figure 3).

After 21 days in adipogenic media, both hACL-SCs and hMCL-SCs expressed high levels of PPARγ and LPL (lipoprotein lipase), two adipogenesis markers, indicating that the cells had differentiated into adipocytes (Figure 7A). When grown in chondrogenic media, these ligament stem cells differentiated into chondrocytes, evidenced by upregulation of Sox-9 and Collagen type II expression (Figure 7B), which are two markers for chondrogenesis. Finally, both hACL-SCs and hMCL-SCs in osteogenic media differentiated into osteocytes, as two osteogenesis markers Runx2 and ALP were significantly upregulated (Figure 7C).

Using respective histochemical staining, we further demonstrated that both hACL-SCs and hMCL-SCs differentiated into adipocytes, chondrocytes, and osteocytes in the respective induction medium, evidenced by formation of lipid droplets (Figure 8A), glycosaminoglycans (GAG)-rich matrix (Figure 8C), and calcium-rich deposits (Figure 8E). Of note is that these stem cells could form cartilage-like pellets in chondrogenic induction medium (insets, Figure 8C). Semi-quantification of stained areas showed that there were significant differences in the extent of adipogenesis (Figure 8B), chondrogenesis (Figure 8D), and osteogenesis (Figure 8F) between hACL-SCs and hMCL-SCs.