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.

After informed consent of parents, 15 - 20 cm of umbilical cord close to the placenta were cut and immediately squeezed in order to drain the extra blood. The umbilical cord was placed into 100 - 200 ml transport medium including DMEM-HG (Gibco, cat; 12800 - 058) supplemented with 2 mM L-Glutamine, 3% (w/v) Pen/strep (Gibco cat; 15140) and 5 μg/ml amphotericin (sigma cat; A2942) in sterile glass jar with lid and transferred to laboratory at 4˚C. The umbilical cord was washed in PBS (Phosphate Buffer Saline) and cut into smaller pieces (3 - 5 cm). Amniotic membrane was removed carefully by making a longitudinal incision in each piece and by using a forceps, blood vessels were removed. At the end of this stage, the remaining tissue is Wharton’s jelly (WJ). WJ tissue was cut into small pieces, approximately 3 - 5 mm in size and 10 - 20 mg in weight. Border of each pieces of explants were carefully trimmed and washed twice in PBS. 6 - 7 explants pieces transferred to each 25 cm² culture flask and incubated 5 min. To achieve a better attachment, 3 ml pre warmed DMEM/HG supplemented with 15% FBS (Fetal Bovine Serum) (Gibco cat; 41G9863K) and L-Glutamine (2 mM) added to each flask as medium were lower the top of pieces. In addition, to prevent shaking that may lead to float of explants pieces the primary culture were incubated at 37˚C, 5% CO₂ for 72 h and after that medium were changed carefully. During 5 - 7 days later, explants buds were observed around pieces (Figure 1). Every other day, the

cells were fed with fresh medium for 5 to 8 days following cell migration. The explants pieces were picked up and allowed the cells to achieve more confluence for 3 - 5 days (Figure 1). Migrated cells detached with prewarmed Trypsin-EDTA (Gibco cat; 25300) solution and by adding 6 mL fresh medium (DMEM/HG with 15% FBS) to each flask, Trypsin-EDTA solution was neutralized and cell distribution was homogenized (in situ passage; P₀, Figure 2(A)). 24 hours later the medium should be changed. Next passages took place at 80% - 90% cell confluence.

HUC-MSCs were grown in six-well plates at a density of 3000 cell/cm² in DMEM-HG media until reaching 90% confluence. The medium was changed with adipogenic induction medium for 21 days. Adipogenic medium was composed of 50 µg/ml L-ascorbic acide-2-phosphate (sigma, cat; A8960), 100 nM dexametazon (sigma, cat; D4902) and 50 µg/ml indometacin (sigma, cat; I7378) [15] . The adipogenic induction medium was changed every 3 days. After 3 weeks to evaluate the formation of Lipid vacuoles, Oil Red O (Bio-IDEA cat; 03111) staining was used as follow: all induction media was aspirated, each well was washed with 2 ml PBS after and 2 ml of 10% Formalin was added to each well then incubated for 25 min at room temperature in order to cell fixation. After 25 mins the Formalin was aspirated and 2 ml of Oil Red O working solution was added to each well and after 5 mins, cells were washed indirectly with tap water. Cells were observed using an inverted phase contrast microscope and Lipid-vacuoles were red (Figure 3). As a control sample equal number of cells were cultured in DMEM-HG supplemented with 10% FBS for 21 days.

HUC-MSCs were differentiated toward the osteogenic linage according to the protocol described by Wang et al. [15] . Mesenchymal stem cells in P₃ were cultured in six-well plates at a density of 3000 cells/cm² in DMEM- HG media until reaching 90% confluence. Osteogenic medium was added to cells for 21 days. Osteogenic induction medium consisted of 10 nM Dexamethazone, 10 nM β-glycerophosphate (Sigma cat; 50,020), 0.05 nM L-ascorbic acid-2-phosphates and 10% FBS in DMEM-HG. As a control sample equal number of cells were cultured in DMEM-HG supplemented with 10% FBS for 21 days. The media were replaced every 4 days in both groups. At the end of 21 days, the cells were stained by Alizarin Red S (sigma cat; A5533) for assessment of calcium deposition and observed under a phase contrast inverted microscope (Figure 3).

The isolated HUC-MSCs were trypsinized in passage 3 and transferred into 15ml Falcon tube. By adding 2 - 3 ml PBS, cells were centrifuged (5 min, 2000 rpm). The supernatant was aspirated and PBS supplemented with 5% FBS added to cell plate and resuspended. The cells were incubated for 30 min at 4^◦C with 5 µl anti-human antibodies against CD105 (PE, 12-1057-41), CD90 (FITC, 11-0909-41), CD34 (FITC, 11-0349-41) and CD45 (FITC, 11-9459-41) in dark. All antibodies were purchased from ebioscience. Finally, 1 - 2 ml PBS containing 5% FBS was added to samples and centrifuged (4˚C, 5 min, 2000 rpm). By adding 1ml PBS to plates and resusspend the cells samples were assessed using the Dako Galaxy flow cytometry and the data were analyzed using Flowjo software (Figure 4).

HUC-MSCs were cultured in 25 cm² flasks and allowed to reach 80% - 90% confluence in DMEM-HG medium supplemented with 10% FBS (v/v). At the first stage, cells differentiated into definitive endoderm by adding activinA (100 ng/ml) (sigma, cat; A4941) and wnt-3α (25 ng/ml) (sigma, cat; SRP3259) into RPMI medium (Gibco, cat; 51,800 - 019) supplemented with BSA (0.1% v/v) for 3 days. At the second stage, the cells were washed with phosphate-buffered saline (PBS). One group (group A) was treated with bFGF (64 ng/ml) (Millipore, cat; GF003) for 5 days. Second group (group B) was treated with cyclopamine-KADD (0.25 µM) (CALBIOCHEM, cat; 299,804) for 5 days. Third group (group C) was treated with bFGF (64 ng/ml) and cyclopamine-KAAD for 5 days. Forth group (group D) was untreated as control. For all groups the medium was also changed from RPMI into DMEM-HG (Gibco). All details about culture condition described in Table 1.

At the end of differentiation period (after second stage), cells were tripsinized and collected. Using RNA spin Mini RNA Isolation Kits (Qiagen) total RNA were extracted according to manufacturer’s instructions. The total RNA was converted to first strand of cDNA using cDNA synthesis kit (Fermentas, cat; K1622) and oligo (dT) as primers. Real-time PCR assays were performed in douplicat. GAPDH was used as housekeeping control gene. The sequences of primers used for Real-time PCR are listed in Table 2. Power syber green PCR master mix (ABI, cat; 4367659) was used according to manufacturer’s instructions. Thermo-cycles condition was: 95˚C for 10 mins followed by 50 cycles at 95˚C for 10 seconds, 56˚C for 30 seconds and 72˚C for 30 seconds. A non- template control was used as a negative control for each gene. Data were analyzed using Livak method (also known as the 2^−ΔΔCt method).

Analysis of VAriance (ANOVA) was used to examine the statistical significance of the differences observed among the treatments. Statistical analysis was performed using SPSS version 14 software (Chicago, IL).

Explants technique was used to prevent damage caused by mechanical and enzymatic methods. After 5-8 days, cell buds were observed around explants (Figure 1). These cells were adherent. In the following days, the cell morphology was changed from round to monolayer spindle-like shape with a whir pool-like arrangement. We found that after explants pieces discard at 14th day, the cells should be passages in situ to keep cell density and uniform cell distribution. It should be mentioned; our observation showed that cell-cell interaction is so important to maintain cell dividing and viability because in low cell density proliferation and viability decreased (data not shown).

The cells treated with adipogenic induction medium for 21 days, showed a lot of lipid vacuoles that stained by Oil Red O (Figure 3(A)). While in osteogenic induction medium, deposition of calcium stained as red color by Alizarin Red (Figure 3(B)). In non-treated control cells, no mineralized matrix and lipid vacuole were observed (Figure 3(C) & Figure 3(D)).

Flow cytometric analysis indicated high level expression of mesenchymal cell surface markers (CD 105 and CD 90) but not expression of hematopoietic markers (CD34, CD45) in the mesenchymal stem cells of Wharton’s jelly.

Real-time analysis showed that Foxa2 and Sox17 were upregulated in cells when treated with activin A and wnt3-α (data not shown). Our results also showed that Pdx-1 had peak expression in cells treated with bFGF and cyclopamine in combination, in comparison with other groups (Figure 5).