Phosphate-buffered saline (PBS), fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), antibiotic (penicillin–streptomycin), alginate and trypsin, HEPES, L-glutamine, Collagenase Type I, Hyaluronidase, trypsin-EDTA, NaCl and CaCl₂ were purchased from Sigma-Aldrich (MO, USA). All chemicals were used directly without further purification. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted.

The study protocol was approved by the Ethics Committee of Ahvaz Jundishapour University of Medical Sciences (AJUMS) and all procedures were performed according to ethical committee approval. UC cells were harvested from the umbilical cords of healthy full-term infants born by cesarean section (C/S). Before collection of UC informed consent received from the mothers.

Protocol design for this study is presented in Figure 1. According to this protocol, the work was divided into five steps; isolation phases (by explant or mixed enzymatic-explant procedures), proliferation of MSCs, flowcytometry with negative and positive surface markers, two dimensional and three dimensional cultures and evaluation of MSCs (Figure 1).

All of UC were collected from Obstetrics Department of Ahvaz Jundishapour University of Medical Sciences (AJUMS) and approximately, 20 cm length pieces cut from each UC. Immediately after C/S, the cords were transferred to sterile containers containing sterile transport medium (DMEM (low glucose) containing 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B. and stored at 4˚C for 2 - 6 hrs before tissue processing. Collected UC were transported to laboratory of Cellular and Molecular Research Center (CMRC), Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapour University of Medical Sciences (AJUMS).All the procedure of collection and transport of C/S were performed under sterile conditions.

Isolation and propagation of MSCs from above small pieces from WJ were carried out according to the protocol is found elsewhere. At least two investigators cooperated in tissue processing and Wharton’s jelly dissection. MSCs isolation were performed in cold, sterile dissection solution consisting of 1% antibiotic solution in PBS to give 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B. Briefly, in laminar hood and under sterile conditions, each piece of umbilical cord was cut into smaller 2 - 4-cm length pieces. The cord was washed gently with carrying solution three times and squeezed with curved forceps to remove trapped blood within the umbilical blood vessels. The washing was repeated until remove any blood or blood clots. At the external surface of each piece, horizontal sections are engraved with a sterile scalpel then cut open lengthwise

with sterile scissors and forceps and cut the outer sheath of UC sections to extract its inner surface containing the WJ. Then, the exposed vein and arteries were removed by pulling with forceps and peeled away from the WJ. Extracted WJ was cut into 3 - 5-mm pieces with a scalpel and washed again with sterile dissection solution [12-15] (Figure 2).

Cells were allowed to adhere to culture flask and nonadherent cells were removed by changing the medium. When the adherent cells reached confluence, covering 70% - 80% of culture flask, the cells were washed once with PBS and trypsinized with 0.25% trypsin-EDTA. After detachment of the adherent cells, suspension centrifuged at 200 - 300 g for 10 min, supernatant discarded, cell pellets washed in medium. Then were resuspended

in medium and transferred to new culture flask at a density 250 × 10³ cells/cm². During subculture, they were passaged every 5 days; medium was replaced every 3 days. Expansion medium consisted of DMEM (low glucose) with 2 mM L-glutamine, supplemented with 20% fetal bovine serum, 100 U penicillin/streptomycin. Adherent cells were cultured until they reached 80% - 90% confluence [12-15]. For further passaging above procedures were conducted again.

The MSCs cultures were routinely visualized using an inverted research microscope IX50 (Olympus, Japan) and the phenotype and growth of primary and passaged cells were monitored Photomicrographs were taken with a digital camera model of Cyber-shot DSC-HX9V (Sony, Japan) under inverted phase-contrast optics.

MSCs were analyzed for surface cell markers by Flow cytometry as described in the Supplemental Data. Cells were trypsinized. And total of 5 × 10⁵ cells were resuspended in PBS and 5% FBS incubated with phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated antibodies against CD73, CD31 and IgG (ebioscince, USA) for 30 min at dark room at 4˚C. Excess antibody was removed by washing and detection of PE and FITC labeling was accomplished on a Dako Glaxy flocytometer. Mouse isotype-matched Antibody (Ab) were used as controls (ebioscince, USA) [12-15].

For alginate hydrogel scaffolding, alginate of 2.0% w/v were prepared as previously described by adding alginic acid sodium salt from brown algae, into 0.15 M NaCl, and 0.025 M HEPES in deionized water by stirring. The solution was then sterilized via 0.45-nm filter [14].

Isolated MSCs (1× 10⁶ cells/ml) were resuspended in 2 - 5 ml prepared hydrogel and then slowly expressed through a 22 gauge needle into a 102 mM CaCl₂ solution. The hydrogels containing encapsulated hMSC were allowed to polymerize for a period of 10 minutes in the CaCl₂ solution. Next, microcapsules were washed 2 - 3 times 10 volumes of 0.15 M NaCl. The alginate/MSCs constructs were finally placed in culture flask with complete culture medium composed of DMEM (low glucose) with 2 mM L-glutamine, supplemented with 20% fetal bovine serum, 100 U penicillin/streptomycin and with controlled environmental conditions (37˚C, 5% CO₂, 95% O₂ and 95% humidity) [10,11].

In the end of MSCs 3D culture in hydrogel scaffold, the constructs were fixed in Bouin’s fixative solution, dehydrated through a series of graded alcohols, cleared with xylene, and embedded in paraffin wax. Five to sevenmicrometer sections were cut, and then stained with hematoxylin & eosin.

Data were given as mean ± standard deviation and were statistically analyzed. The difference between the mean was considered significant when p < 0.05.

Cells with a MSCs phenotype were isolated from mixed enzymatic-explant within 24 - 48 h of collection, while in explant procedure through 7 - 10 days after collection, MSCs phenotype cells were isolated. Therefore, when compared isolation of MSC from explant culture was much slower to the enzymatic-explant cultures (2 ± 8.7 days compared to 15 ± 9.9 days, respectively) which led to confluence was reached much slower compared to the same enzymatic cultures. Although cell growth increased with time in enzymatic-explant culture, the increases were not as high as for with explants culture. Also enzymatic cultures produced MSCs at high efficiency (Figures 3 and 4).

There was no difference between explant and a mixed enzymatic-explant culture in proliferation after initial cell isolation either 24 - 48 hrs. In enzymatic-explant culture or 7 - 10 days in explant culture. Both of procedures were shown to form clumps and colonies with dense centers. These colonized populations were observed at the early stages of culture. Cells were cultured and maintained until passage 7 - 8 (Figures 3 and 4).

Phenotypic changes gradually appeared as round cell in UC pieces into homogeneous spindle-shape and typical fibroblast-like shape cells which continued to proliferate. When these spindle-shaped cells appeared in culture, colonies formed thereafter. Although hMSC maintained their characteristics such as the fibroblastic morphology over the passages, primary and first-passage cultures displayed an epithelioid or short fibroblastic-like phenoltype but in further passages, cell transformed into long spindle-shaped fibroblast-like cells (Figure 5).

The immunophenotype of the WJSC was similar in both enzymztic and explant and cultured stem cells were positive for general mesenchymal markers such, CD73, but negative against hematopoietic markers, CD31 (Figure 1) although there were differences in growth rates. Therefore these data suggested that the UC derived cell is one of the MSCs populations similar to those from bone marrow or adipose tissue (Figure 6).

Homogenous alginate beads successfully produced. Morphology of viable MSCs in the beads did not significantly revealed a different morphology pattern before and after the bead formation process. They had round shaped similar to round shape of stem cells in UC.

After 7 - 10 days of culture, the microscopic pictures suggested formation of three-dimensional cell aggregates

as mounds, and the mounds in 3D were larger in size and greater in numbers compared with 2D (Figures 5 and 7).

Typical histological appearance of sections revealed evidence of round cells with clear cytoplasm and nucleus which aggregated in some part of scaffold.