Umbilical cord mesenchymal stem cells (cord stem cells) were obtained from Dr. Biagio Saitta at Cedars-Sinai Medical Center/Regenerative Medicine Institute, Las Angeles, CA, and were isolated as previously reported [20]. Briefly, cells were harvested at birth from the placenta of full-term deliveries with the consent of the parents. This was done under the International Review Board-approved protocol to the New Jersey Cord Blood Bank, where Saitta previously worked. Blood was collected within 10 min of placenta removal and was drained into a plastic bag with 25 mL of citrate phosphate dextrose anticoagulant solution (Medsep Corporation; Covina, CA). Cells were processed as reported previously [20] and were used between passages 5–10. Detailed characterization and expression markers can be found in Markov et al.'s manuscript [20].

The cord stem cells were plated on transwell 6-well plates containing 24-mm polycarbonate membrane inserts with 0.4 μm pores (Costar; Charlotte, NC) at a density of 2 × 10⁶ cells in 1.5 mL EMEM. The seeding density was determined by a number of preliminary experiments where the amount of extracellular matrix deposited was the critical element. Constructs were allowed to grow for 4 weeks in either VitC media—EMEM with 10% FBS and 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (VitC: Wako Chemicals USA, Inc.; Richmond, V.A.)—only (control), or VitC media + 0.1 ng/mL TGF-β1 (TGF-β1 treated). Preliminary concentration-dependent experiments with TGF-β1 ruled out higher concentrations of the growth factor due to construct contraction. At week 4, samples of the resulting constructs were collected and processed for immunofluorescence microscopy, transmission electron microscopy (TEM) and Real Time RT-PCR.

Constructs were fixed in 4% paraformaldehyde and processed for immunofluorescence, as previously described [21,22]. Constructs were stained with phalloidin-rhodamine (Invitrogen; Carlsbad, CA, USA), which binds and stains the f-actin filaments and allows for the visualization of all cells within the construct. Also, TOPRO-3 iodide (Invitrogen) was used to stain the cell nuclei. The constructs were imaged on a TCS-SP2 confocal microscope (Leica Microsystems; Bannockburn, I.L., UK). The total thickness of the constructs was calculated using the confocal z-series, where we measured from the very top cell to the very bottom one. To obtain a visual confirmation of this measurement, orthogonal sections of the same confocal z-series were created with the Image Pro Plus software.

At 4 weeks, the constructs were fixed in ½ strength Karnovsky's fixative (2% paraformaldehyde, 2.5% glutaraldehyde in cacodylate buffer, pH 7.4) and processed, as previously described [23]. Briefly, samples were cut transversely to the plane of the construct, using a diamond knife ultramicrotome (LKB ultramicrotome; Bromma, Sweden). Sections (60–90 Å) were obtained, examined and photographed with a Philips 410 Transmission Electron Microscope (Philips Electronics N.V.; Eindhoven, The Netherlands) equipped with a digital camera.

Using Image Pro Plus (v.7: Media Cybernetics; Bethesda, MD, USA), we quantified the total cell number per construct per condition, as well as, cell per unit volume. A minimum of 3 confocal z-series was used for each condition, and their average was plotted and analyzed.

Total RNA was extracted using the RNeasy kit (Qiagen; Valencia, CA, USA). Genomic DNA was removed by incubation with RNase-free DNase I (M0303S: New England BioLabs; Ipswich, MA, UK) in the presence of RNase inhibitor. The RNA was annealed with random hexamer and oligo-dt primers, and first strand synthesis was carried out with MuLV reverse transcriptase. Negative controls were performed without reverse transcriptase. Real-Time PCR was performed on an Applied Biosystems 7300 Real-Time PCR system (Foster City, CA, USA) using ABI TaqMan human gene expression assays for perlecan (Hs01078536.m1), keratocan (Hs00559942.m1), decorin (Hs00370384.m1), lumican (Hs00158940.m1), collagen 1A1 (Hs00164004.m1), collagen 5A1 (Hs00619088.m1), and the eukaryotic 18S rRNA endogenous control (4308329), which was used for normalization. The cycling parameters were as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Calculations were carried out using the δδCt method of relative quantitation. Results were expressed as the mean of relative mRNA levels ± SEM.

Cuprolinic blue is an electron-dense reagent which has been shown to identify sulfated GAGs and to demonstrate the association of sulfated GAGs with matrix molecules, such as collagen [24]. The length of cuprolinic blue stained filaments indicates the extent of their sulfation. Briefly, the samples were stained with 0.2% Cuprolinic blue (in buffer containing 0.3 M MgCl₂) for 24 hours, dehydrated and processed for electron microscopy [25-27].

One hundred cuprolinic blue stained GAG side chains were randomly chosen from each region—apical, middle and basal—of each construct, and their length were measured using ImageJ v.1.5 [28]. Results were analyzed for significance (p < 0.05) and plotted (Prism v.5.0: GraphPad Software, Inc.; La Jolla, CA, USA) [27].

Fibril diameters for each condition were measured and compared using Photoshop (v.10.0.1). The scale bar for the TEM was used to calibrate the measurements. At least ten randomly chosen electron micrographs were used for each condition and a total of more than 100 fibrils were measured. Results were plotted and analyzed for significance (p < 0.05).

All experiments were repeated at least three times and data was analyzed for significant variations (p < 0.05) using the Student's t-test and Dunnett's Multiple Comparison test.

In our previous work [21,22,29], we have shown that corneal fibroblasts have the ability to secrete and lay down a fibrous collagen extracellular matrix in the presence of derivatized VitC. We have demonstrated the effect of TGF-β1, -β2 and -β3 in these cultures [22]. In the current study, we examined the ability of cord stem cells to synthesize a matrix in the presence of VitC ± TGF-β1. The development of an extracellular matrix in vitro would be useful for many applications in stem cell tissue engineering.

After 4 weeks, cord stem cells cultured in the presence of VitC resulted in a construct approximately 35 um thick, which is similar to constructs synthesized by human corneal fibroblasts under the same conditions (Figure 1.1 and 1.2A, C) [21,22]. Surprisingly, when TGF-β1 was added to the cord stem cell cultures, no significant increase was found in the construct thickness (Figure 1.1 and 1.2D). This is in contrast to constructs synthesized by human corneal fibroblasts, where the thickness increased 2–3 fold (p < 0.0001; Figure 1.1 and 1.2A, B). Results were confirmed and shown in Figure 1.2 using orthogonal sections created with Image Pro Plus software. This data suggests that a differential path of expression may exist between the two cell types.

Cell-matrix interactions revealed similarities between the two cell lineages. Cord stem cells assembled aligned collagen that alternated directions in Controls (Figure 2A). When stimulated with TGF-β1 (Figure 2B), the number of alternating layers appeared to decrease resulting in a higher degree of alignment. This observation was similar to what was seen previously with corneal fibroblasts [21]. Also, in the cord stem cell constructs, cells were elongated and very similar in morphology to human corneal fibroblasts (data not shown), with the addition of TGF-β1, these cells resulted in more aligned actin-like filaments, indicative of myofibroblasts (data not shown). Vesicles were also seen under both conditions (Figure 2); however, they were more abundant when constructs were treated with TGF-β1 (Figure 2B). Vesicles were recently identified by us and reported as a potential myofibroblast marker [22].

For each sample, the cell number and the resulting cell number per unit volume were calculated using Image Pro Plus. When cells were stimulated with TGF-β1, human corneal fibroblast constructs had a higher total number of cells (∼3 fold) compared to cord stem cell constructs (p < 0.0001; Figure 3A). However, there was no difference in the control groups for either lineage. Taking into account that the control construct thickness for both cell lineages was approximately equal, and only the thickness of the human corneal fibroblast construct increased with the addition of TGF-β1 (Figure 1), we calculated the cell number per unit volume for all conditions. The data showed that there was no difference in the cell number per unit volume between control and TGF-β1 stimulated cord stem cell constructs (Figure 3B). However, human corneal fibroblast constructs increased in cell number per unit volume upon TGF-β1 stimulation (Figure 3B). Human corneal fibroblasts showed significantly higher numbers of cells per unit volume, both in Controls and TGF-β1 when compared to cord stem cells (p < 0.001). Overall our data indicates that the cord stem cells assembled a more extensive matrix per cell than the human corneal fibroblasts.

In our previous work with human corneal fibroblasts [22], we found that the Control and TGF-β1 treated construct fibril diameter means were 27 and 32 nm, respectively, which was similar to that seen in the mature human corneal stroma, 30–35 nm (hydrated type I/V collagen). In the present study with the cord stem cells, the fibril diameters in the Control constructs (Figure 4A) had a range of 15–42 nm, with a mean diameter of 28 nm. In response to TGF-β1 (Figure 4B), the mean fibril diameter was 32 nm, with a range of 23–60 nm. Thus, TGF-β1 slightly, but significantly (p < 0.001), increased the mean collagen fibril diameter of the cord stem cells. The data indicates that the fibril diameters of the cord stem cells were similar to those seen in the human corneal fibroblast constructs and the mature human corneal stroma. Of note, following injury in human corneal stroma, the fibril diameter range was found to increase (15–65 nm) [30].

As Type I and V collagen are known to be the major stromal collagens, we examined their expression in the construct to observe if their expression was modulated by cord stem cells in the presence of TGF-β1 (Figure 5). Collagen I (Figure 5A) mRNA was only enhanced in the constructs synthesized by human corneal fibroblasts (p < 0.005). We found that TGF-β1 enhanced the Collagen V mRNA expression (Figure 5B) in both cell lineages (p < 0.05), indicating that the constructs both showed a response to changes in extracellular matrix independent of the cell/matrix density.

Proteoglycans are known to play an important role in extracellular matrix organization in development and during pathology. Previously, in human corneal fibroblast 3D construct, the expression of proteoglycan cores (lumican, decorin and keratocan) was detected. In addition, the presence of associated sulfated GAGs was examined throughout the depth of the construct [27]. Since the regulation of proteoglycans may be critical for the design of a cornea-like construct, one of the goals of this study was to examine the expression of proteoglycan transcripts in the cord stem cell constructs.

The expression of proteoglycan core transcripts was performed using Real Time RT-PCR. In the cord stem cell constructs, there was a ∼2-fold decrease in decorin and keratocan mRNA (p < 0.05) when the cultures were incubated in the presence of TGF-β1 (Figure 6A). While there was no detectable change in lumican mRNA, there was a significant increase (p < 0.05) in perlecan mRNA (Figure 6A). This regulation was similar to what was seen in human corneal fibroblasts (Figure 6B). Upon TGF-β1 treatment, keratocan was downregulated ∼3-fold (p < 0.05), lumican remained unchanged, and decorin and perlecan both were upregulated.

The major change in response to TGF-β1 in the cord stem cell constructs may be the significant increase in sulfated GAGs. The presence of GAGs was analyzed throughout the construct using Cuprolinic Blue, which binds to sulfated moieties [24,27]. The GAG filaments were detected in either the plane of the collagen fibril (Figure 7.1B and D, “GAG” arrows) or associated with fibrils out of the plane of the image. In control constructs of both human corneal fibroblasts (Figure 7.1A) and cord stem cells (Figure 7.1C) the filaments are present and are ∼20 nm and 30 nm, respectively (Figure 7.2). Upon the addition of TGF-β1, the GAG filament length in the human corneal fibroblast (Figure 7.1B) and cord stem cell (Figure 7.1D) constructs increased significantly (p < 0.0001, Figure 7.2), indicating a similar mechanism of extracellular matrix organization.