Limits...
Angiogenic Potential of Human Neonatal Foreskin Stromal Cells in the Chick Embryo Chorioallantoic Membrane Model.

Vishnubalaji R, Atteya M, Al-Nbaheen M, Oreffo RO, Aldahmash A, Alajez NM - Stem Cells Int (2015)

Bottom Line: When hNSSCs were seeded onto the top of the CAM, human von Willebrand factor (hVWF), CD31, smooth muscle actin (SMA), and factor XIIIa positive cells were observed in the chick endothelium.Interestingly, undifferentiated hNSSCs showed a propensity to differentiate towards ectoderm with indication of epidermal formation with cells positive for CD1a, CK5/6, CK19, FXIIIa, and S-100 cells, which warrant further investigation.Our findings imply a potential angiogenic role for hNSSCs ex vivo in the differentiated and undifferentiated state, with potential contribution to blood vessel formation and potential application in tissue regeneration and vascularization.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia.

ABSTRACT
Several studies have demonstrated the multipotentiality of human neonatal foreskin stromal cells (hNSSCs) as being able to differentiate into adipocytes and osteoblasts and potentially other cell types. Recently, we demonstrated that hNSSCs play a role during in vitro angiogenesis and appear to possess a capacity to differentiate into endothelial-like cells; however, their angiogenic potential within an ex vivo environment remains unclear. Current study shows hNSSCs to display significant migration potential in the undifferentiated state and high responsiveness in the in vitro wound healing scratch assay. When hNSSCs were seeded onto the top of the CAM, human von Willebrand factor (hVWF), CD31, smooth muscle actin (SMA), and factor XIIIa positive cells were observed in the chick endothelium. CAMs transplanted with endothelial-differentiated hNSSCs displayed a higher number of blood vessels containing hNSSCs compared to CAMs transplanted with undifferentiated hNSSCs. Interestingly, undifferentiated hNSSCs showed a propensity to differentiate towards ectoderm with indication of epidermal formation with cells positive for CD1a, CK5/6, CK19, FXIIIa, and S-100 cells, which warrant further investigation. Our findings imply a potential angiogenic role for hNSSCs ex vivo in the differentiated and undifferentiated state, with potential contribution to blood vessel formation and potential application in tissue regeneration and vascularization.

No MeSH data available.


Related in: MedlinePlus

Explant organ culture system of neonatal foreskin. (a) Cell migration from skin tissue after 3 days (left); fibroblast-like cells can be observed migrating and sprouting out from tissue after 12 days (right). (b) A transwell migration assay where cells migrated to the lower chamber visualized by Eosin staining. The transwell migration assay was repeated and the total number of cells that migrated was quantified to determine the relative numbers of migrated cells (b, lower panel). Data are presented as mean ± S.D. The experiment was run in triplicate. (c) The real time migration was executed using the xCELLigence RTCA DP device. Cells were seeded per well in 16-well microelectronic sensing, two-chamber transwell plates (CIM-plates). The electrical impedance was captured every 15 min for an experimental duration of ~68 h. The rate of migration is expressed as the CI or the change in electrical impedance at each time-point (c, left). Values are expressed as the ±SEM of the 8 replica wells from three independent experiments (c, right). (d) Measurement of undifferentiated hNSSCs cell migration by in vitro wound healing scratch assay. The migration ability was assessed 12 h and 24 h from injury. The figure shows the migration of undifferentiated cells immediately after the scratch 0 h, 12 h, and 24 h (Bar = 100 μm). (e) ALDH activity of hNSSCs was measured using the Aldefluor assay and FACS analysis. Cells incubated with specific inhibitor of ALDH, DEAB, were used to establish the baseline fluorescence of these cells and to define the Aldefluor positive region.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4499640&req=5

fig1: Explant organ culture system of neonatal foreskin. (a) Cell migration from skin tissue after 3 days (left); fibroblast-like cells can be observed migrating and sprouting out from tissue after 12 days (right). (b) A transwell migration assay where cells migrated to the lower chamber visualized by Eosin staining. The transwell migration assay was repeated and the total number of cells that migrated was quantified to determine the relative numbers of migrated cells (b, lower panel). Data are presented as mean ± S.D. The experiment was run in triplicate. (c) The real time migration was executed using the xCELLigence RTCA DP device. Cells were seeded per well in 16-well microelectronic sensing, two-chamber transwell plates (CIM-plates). The electrical impedance was captured every 15 min for an experimental duration of ~68 h. The rate of migration is expressed as the CI or the change in electrical impedance at each time-point (c, left). Values are expressed as the ±SEM of the 8 replica wells from three independent experiments (c, right). (d) Measurement of undifferentiated hNSSCs cell migration by in vitro wound healing scratch assay. The migration ability was assessed 12 h and 24 h from injury. The figure shows the migration of undifferentiated cells immediately after the scratch 0 h, 12 h, and 24 h (Bar = 100 μm). (e) ALDH activity of hNSSCs was measured using the Aldefluor assay and FACS analysis. Cells incubated with specific inhibitor of ALDH, DEAB, were used to establish the baseline fluorescence of these cells and to define the Aldefluor positive region.

Mentions: hNSSCs were isolated and cultured in accordance with our previously published protocols [6, 8]. In brief, cells were isolated by explant organ culture to establish outgrowth cell culture (Figure 1(a)). Newborn foreskins were received from voluntary circumcisions with informed consent. Tissues were washed and the epidermis was removed followed by the dermis. Tissues were placed in culture dishes with the epidermis layer facing upwards and the dermis area in contact with the plastic surface with a droplet of culture medium. Cultures were maintained at 37°C and 5% CO2 in a humidified environment. Additional media were added following cell attachment and culture was maintained for 7 days or until outgrowths of fibroblast-like spindle shaped cells were observed. At 70–80% confluency, cells were trypsinized and residual tissues were removed. The culture medium consisted of Dulbecco's Modified Eagle Medium (DMEM) supplemented with D-glucose 4500 mg/L, 4 mM L-Glutamine, and 110 mg/L Sodium Pyruvate, 10% Fetal Bovine Serum (FBS), 1x penicillin-streptomycin (Pen-strep), and nonessential amino acids (all purchased from Gibco-Invitrogen, USA).


Angiogenic Potential of Human Neonatal Foreskin Stromal Cells in the Chick Embryo Chorioallantoic Membrane Model.

Vishnubalaji R, Atteya M, Al-Nbaheen M, Oreffo RO, Aldahmash A, Alajez NM - Stem Cells Int (2015)

Explant organ culture system of neonatal foreskin. (a) Cell migration from skin tissue after 3 days (left); fibroblast-like cells can be observed migrating and sprouting out from tissue after 12 days (right). (b) A transwell migration assay where cells migrated to the lower chamber visualized by Eosin staining. The transwell migration assay was repeated and the total number of cells that migrated was quantified to determine the relative numbers of migrated cells (b, lower panel). Data are presented as mean ± S.D. The experiment was run in triplicate. (c) The real time migration was executed using the xCELLigence RTCA DP device. Cells were seeded per well in 16-well microelectronic sensing, two-chamber transwell plates (CIM-plates). The electrical impedance was captured every 15 min for an experimental duration of ~68 h. The rate of migration is expressed as the CI or the change in electrical impedance at each time-point (c, left). Values are expressed as the ±SEM of the 8 replica wells from three independent experiments (c, right). (d) Measurement of undifferentiated hNSSCs cell migration by in vitro wound healing scratch assay. The migration ability was assessed 12 h and 24 h from injury. The figure shows the migration of undifferentiated cells immediately after the scratch 0 h, 12 h, and 24 h (Bar = 100 μm). (e) ALDH activity of hNSSCs was measured using the Aldefluor assay and FACS analysis. Cells incubated with specific inhibitor of ALDH, DEAB, were used to establish the baseline fluorescence of these cells and to define the Aldefluor positive region.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4499640&req=5

fig1: Explant organ culture system of neonatal foreskin. (a) Cell migration from skin tissue after 3 days (left); fibroblast-like cells can be observed migrating and sprouting out from tissue after 12 days (right). (b) A transwell migration assay where cells migrated to the lower chamber visualized by Eosin staining. The transwell migration assay was repeated and the total number of cells that migrated was quantified to determine the relative numbers of migrated cells (b, lower panel). Data are presented as mean ± S.D. The experiment was run in triplicate. (c) The real time migration was executed using the xCELLigence RTCA DP device. Cells were seeded per well in 16-well microelectronic sensing, two-chamber transwell plates (CIM-plates). The electrical impedance was captured every 15 min for an experimental duration of ~68 h. The rate of migration is expressed as the CI or the change in electrical impedance at each time-point (c, left). Values are expressed as the ±SEM of the 8 replica wells from three independent experiments (c, right). (d) Measurement of undifferentiated hNSSCs cell migration by in vitro wound healing scratch assay. The migration ability was assessed 12 h and 24 h from injury. The figure shows the migration of undifferentiated cells immediately after the scratch 0 h, 12 h, and 24 h (Bar = 100 μm). (e) ALDH activity of hNSSCs was measured using the Aldefluor assay and FACS analysis. Cells incubated with specific inhibitor of ALDH, DEAB, were used to establish the baseline fluorescence of these cells and to define the Aldefluor positive region.
Mentions: hNSSCs were isolated and cultured in accordance with our previously published protocols [6, 8]. In brief, cells were isolated by explant organ culture to establish outgrowth cell culture (Figure 1(a)). Newborn foreskins were received from voluntary circumcisions with informed consent. Tissues were washed and the epidermis was removed followed by the dermis. Tissues were placed in culture dishes with the epidermis layer facing upwards and the dermis area in contact with the plastic surface with a droplet of culture medium. Cultures were maintained at 37°C and 5% CO2 in a humidified environment. Additional media were added following cell attachment and culture was maintained for 7 days or until outgrowths of fibroblast-like spindle shaped cells were observed. At 70–80% confluency, cells were trypsinized and residual tissues were removed. The culture medium consisted of Dulbecco's Modified Eagle Medium (DMEM) supplemented with D-glucose 4500 mg/L, 4 mM L-Glutamine, and 110 mg/L Sodium Pyruvate, 10% Fetal Bovine Serum (FBS), 1x penicillin-streptomycin (Pen-strep), and nonessential amino acids (all purchased from Gibco-Invitrogen, USA).

Bottom Line: When hNSSCs were seeded onto the top of the CAM, human von Willebrand factor (hVWF), CD31, smooth muscle actin (SMA), and factor XIIIa positive cells were observed in the chick endothelium.Interestingly, undifferentiated hNSSCs showed a propensity to differentiate towards ectoderm with indication of epidermal formation with cells positive for CD1a, CK5/6, CK19, FXIIIa, and S-100 cells, which warrant further investigation.Our findings imply a potential angiogenic role for hNSSCs ex vivo in the differentiated and undifferentiated state, with potential contribution to blood vessel formation and potential application in tissue regeneration and vascularization.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia.

ABSTRACT
Several studies have demonstrated the multipotentiality of human neonatal foreskin stromal cells (hNSSCs) as being able to differentiate into adipocytes and osteoblasts and potentially other cell types. Recently, we demonstrated that hNSSCs play a role during in vitro angiogenesis and appear to possess a capacity to differentiate into endothelial-like cells; however, their angiogenic potential within an ex vivo environment remains unclear. Current study shows hNSSCs to display significant migration potential in the undifferentiated state and high responsiveness in the in vitro wound healing scratch assay. When hNSSCs were seeded onto the top of the CAM, human von Willebrand factor (hVWF), CD31, smooth muscle actin (SMA), and factor XIIIa positive cells were observed in the chick endothelium. CAMs transplanted with endothelial-differentiated hNSSCs displayed a higher number of blood vessels containing hNSSCs compared to CAMs transplanted with undifferentiated hNSSCs. Interestingly, undifferentiated hNSSCs showed a propensity to differentiate towards ectoderm with indication of epidermal formation with cells positive for CD1a, CK5/6, CK19, FXIIIa, and S-100 cells, which warrant further investigation. Our findings imply a potential angiogenic role for hNSSCs ex vivo in the differentiated and undifferentiated state, with potential contribution to blood vessel formation and potential application in tissue regeneration and vascularization.

No MeSH data available.


Related in: MedlinePlus