We report combination of explants and enzymatic protocol as mixed enzymatic-explant procedure to faster extraction of MSCs from WJ. Umbilical cords (UC) were collected from Imam Khomini Hospital. For explant outgrowth...We report combination of explants and enzymatic protocol as mixed enzymatic-explant procedure to faster extraction of MSCs from WJ. Umbilical cords (UC) were collected from Imam Khomini Hospital. For explant outgrowth, 6 - 9 pieces of WJ were transferred onto tissue culture flask and waited for attachment. For mixed enzymatic-explant, 1 cm3 pieces WJ were placed in enzymatic cocktail comprising 4 mg/ml Collagenase Type I and 1 mg/ml Hyaluronidase and 0.1% trypsin-EDTA. Then isolated cells were analyzed for surface cell markers such as CD73, CD31. Isolated 1.0 × 106 MSCs/ml were encapsulated in alginate hydrogel. Cells with MSCs phenotype were isolated from mixed enzymatic-explant and explant procedures within 24 - 48 hrs and 7 - 10 days, respectively. Both of procedures were shown to form clumps and colonies with dense centers. Phenotypic changes gradually appeared as round cell in UC pieces into homogeneous spindle-shaped and typical fibroblast-like shape cells. By using flow cytometery MSCs showed positive for CD73, and negative for CD31. the morphology of viable MSCs in the beads did not significantly show a different morphology pattern before and after the bead formation process. These findings are indicated that when mixed enzymatic-explant procedure is performed MSCs can be isolated faster and much higher from WJ. These finding is important in comparing with time consuming explants culture for isolation of MSCs.展开更多
Background: Recent studies have focused on generating of insulin-producing cells (IPCs) from pluripotent stem cells. Producing of precursor's population with pancreatic endoderm properties is a challenging issue i...Background: Recent studies have focused on generating of insulin-producing cells (IPCs) from pluripotent stem cells. Producing of precursor's population with pancreatic endoderm properties is a challenging issue in front of regenerative medicine investigators. Previous studies have shown that during pancrease development in lower portion of foregut, signals from notochord suppress sonic hedgehog (Shh) expression and lead to increase expression of pancreatic duodenal homeobox-1 (Pdx-1) as a marker for pancreatic precursor's cells. Therefore, Shh repression is considered as a critical step in IPCs generation protocols. Objective: Isolation and characterization of human umbilical cord mesenchymal stem cells (HUC-MSCs) is the aim of current study. As well as the role of basic fibroblast growth factor (bFGF) alone and in combination with cyclopamine are investigated in creating cells with Pdx-1 expression ability. Methods: Cells differentiate into definitive endoderm by adding activin A and wnt-3α into RPMI medium supplemented with for 3 days. At the second stage, the cells are washed with phosphate-buffered saline (PBS). One group (group A) is treated with bFGF for 5 days. Second group (group B) is treated with cyclopamine-KADD for 5 days. Third group (group C) is treated with bFGF and cyclopamine-KAAD for 5 days. Forth group (group D) is untreated as control. Result: Our results show that bFGF and cyclopamine in combination induce more expression of Pdx-1 in HUC-MSCs.展开更多
文摘We report combination of explants and enzymatic protocol as mixed enzymatic-explant procedure to faster extraction of MSCs from WJ. Umbilical cords (UC) were collected from Imam Khomini Hospital. For explant outgrowth, 6 - 9 pieces of WJ were transferred onto tissue culture flask and waited for attachment. For mixed enzymatic-explant, 1 cm3 pieces WJ were placed in enzymatic cocktail comprising 4 mg/ml Collagenase Type I and 1 mg/ml Hyaluronidase and 0.1% trypsin-EDTA. Then isolated cells were analyzed for surface cell markers such as CD73, CD31. Isolated 1.0 × 106 MSCs/ml were encapsulated in alginate hydrogel. Cells with MSCs phenotype were isolated from mixed enzymatic-explant and explant procedures within 24 - 48 hrs and 7 - 10 days, respectively. Both of procedures were shown to form clumps and colonies with dense centers. Phenotypic changes gradually appeared as round cell in UC pieces into homogeneous spindle-shaped and typical fibroblast-like shape cells. By using flow cytometery MSCs showed positive for CD73, and negative for CD31. the morphology of viable MSCs in the beads did not significantly show a different morphology pattern before and after the bead formation process. These findings are indicated that when mixed enzymatic-explant procedure is performed MSCs can be isolated faster and much higher from WJ. These finding is important in comparing with time consuming explants culture for isolation of MSCs.
文摘Background: Recent studies have focused on generating of insulin-producing cells (IPCs) from pluripotent stem cells. Producing of precursor's population with pancreatic endoderm properties is a challenging issue in front of regenerative medicine investigators. Previous studies have shown that during pancrease development in lower portion of foregut, signals from notochord suppress sonic hedgehog (Shh) expression and lead to increase expression of pancreatic duodenal homeobox-1 (Pdx-1) as a marker for pancreatic precursor's cells. Therefore, Shh repression is considered as a critical step in IPCs generation protocols. Objective: Isolation and characterization of human umbilical cord mesenchymal stem cells (HUC-MSCs) is the aim of current study. As well as the role of basic fibroblast growth factor (bFGF) alone and in combination with cyclopamine are investigated in creating cells with Pdx-1 expression ability. Methods: Cells differentiate into definitive endoderm by adding activin A and wnt-3α into RPMI medium supplemented with for 3 days. At the second stage, the cells are washed with phosphate-buffered saline (PBS). One group (group A) is treated with bFGF for 5 days. Second group (group B) is treated with cyclopamine-KADD for 5 days. Third group (group C) is treated with bFGF and cyclopamine-KAAD for 5 days. Forth group (group D) is untreated as control. Result: Our results show that bFGF and cyclopamine in combination induce more expression of Pdx-1 in HUC-MSCs.
