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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

hNSSCs contribute to neovasculature using ex vivo CAM model. (a) Schematic illustration of the organotypic culture system (liquid-air interface). hNSSCs pellets were placed on the top of a confetti that is in contact with the membrane on the insert. The media diffuse across the insert membrane and confetti to reach the pellet. Microscopic aspect of chick chorioallantoic membrane (CAM) and organotypic cultured hNSSCs with confetti grafted to it. (b) hNSSCs pellet placed on top of a piece of confetti that is in contact with the membrane on the insert. (c) Window (2 cm2) made in the egg shell of a 10-day-old chick embryo with organotypic culture transplanted. (d) After 10 days, implantation shell was opened and blood vessel points were observed; (e and f) note the wheel-spoke pattern of CAM blood vessels around the graft (undifferentiated and differentiated). Stimulation of angiogenesis in CAM by undifferentiated or differentiated hNSSCs. The expansion of new blood vessels was determined by counting branch points after 10 days of cell transplantation. Sprouting and branching vessels are prominent in the CAM transplanted by differentiated hNSSCs compared to the undifferentiated hNSSCs transplantation. (g) Quantification of new blood vessels formed by naive (undifferentiated) and differentiated hNSSCs compared to negative control (confetti without cell transplantation) (n = 5). ∗P < 0.05. (h, i) Paraffin sections of both undifferentiated and differentiated hNSSCs transplanted CAM were stained by haematoxylin and eosin stain to show the vascular density in CAM; blood vessels were counted again (blue colour points indicate vasculature) with Aperio's ImageScope software (Aperio Technologies, Vista, CA, USA) (Bar = 100 μm).
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fig3: hNSSCs contribute to neovasculature using ex vivo CAM model. (a) Schematic illustration of the organotypic culture system (liquid-air interface). hNSSCs pellets were placed on the top of a confetti that is in contact with the membrane on the insert. The media diffuse across the insert membrane and confetti to reach the pellet. Microscopic aspect of chick chorioallantoic membrane (CAM) and organotypic cultured hNSSCs with confetti grafted to it. (b) hNSSCs pellet placed on top of a piece of confetti that is in contact with the membrane on the insert. (c) Window (2 cm2) made in the egg shell of a 10-day-old chick embryo with organotypic culture transplanted. (d) After 10 days, implantation shell was opened and blood vessel points were observed; (e and f) note the wheel-spoke pattern of CAM blood vessels around the graft (undifferentiated and differentiated). Stimulation of angiogenesis in CAM by undifferentiated or differentiated hNSSCs. The expansion of new blood vessels was determined by counting branch points after 10 days of cell transplantation. Sprouting and branching vessels are prominent in the CAM transplanted by differentiated hNSSCs compared to the undifferentiated hNSSCs transplantation. (g) Quantification of new blood vessels formed by naive (undifferentiated) and differentiated hNSSCs compared to negative control (confetti without cell transplantation) (n = 5). ∗P < 0.05. (h, i) Paraffin sections of both undifferentiated and differentiated hNSSCs transplanted CAM were stained by haematoxylin and eosin stain to show the vascular density in CAM; blood vessels were counted again (blue colour points indicate vasculature) with Aperio's ImageScope software (Aperio Technologies, Vista, CA, USA) (Bar = 100 μm).

Mentions: hNSSCs were cultured on the CAM as previously detailed allowing the analysis of hNSSCs in an ex vivo culture (Figure 3) [12]. After Trypsinization, cell pellets were resuspended in 10 mL culture medium (control cells in routine culture medium, induced cells in endothelial induction medium). Cell count was performed to give a cell density of between 3 × 105 and 5 × 105/mL. One mL of cell suspension was added per falcon tube, centrifuged at 400 g for 10 minutes and incubated at 37°C, supplemented with 5% CO2. After 2 days, pellets formed were cultured for 2 more days on organotypic culture until cell attached to the confetti (Figures 3(a) and 3(b)) (hydrophilic PTFE (polytetrafluoroethylene) membranes (0.4 mm pore size); Millipore; UK).


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)

hNSSCs contribute to neovasculature using ex vivo CAM model. (a) Schematic illustration of the organotypic culture system (liquid-air interface). hNSSCs pellets were placed on the top of a confetti that is in contact with the membrane on the insert. The media diffuse across the insert membrane and confetti to reach the pellet. Microscopic aspect of chick chorioallantoic membrane (CAM) and organotypic cultured hNSSCs with confetti grafted to it. (b) hNSSCs pellet placed on top of a piece of confetti that is in contact with the membrane on the insert. (c) Window (2 cm2) made in the egg shell of a 10-day-old chick embryo with organotypic culture transplanted. (d) After 10 days, implantation shell was opened and blood vessel points were observed; (e and f) note the wheel-spoke pattern of CAM blood vessels around the graft (undifferentiated and differentiated). Stimulation of angiogenesis in CAM by undifferentiated or differentiated hNSSCs. The expansion of new blood vessels was determined by counting branch points after 10 days of cell transplantation. Sprouting and branching vessels are prominent in the CAM transplanted by differentiated hNSSCs compared to the undifferentiated hNSSCs transplantation. (g) Quantification of new blood vessels formed by naive (undifferentiated) and differentiated hNSSCs compared to negative control (confetti without cell transplantation) (n = 5). ∗P < 0.05. (h, i) Paraffin sections of both undifferentiated and differentiated hNSSCs transplanted CAM were stained by haematoxylin and eosin stain to show the vascular density in CAM; blood vessels were counted again (blue colour points indicate vasculature) with Aperio's ImageScope software (Aperio Technologies, Vista, CA, USA) (Bar = 100 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: hNSSCs contribute to neovasculature using ex vivo CAM model. (a) Schematic illustration of the organotypic culture system (liquid-air interface). hNSSCs pellets were placed on the top of a confetti that is in contact with the membrane on the insert. The media diffuse across the insert membrane and confetti to reach the pellet. Microscopic aspect of chick chorioallantoic membrane (CAM) and organotypic cultured hNSSCs with confetti grafted to it. (b) hNSSCs pellet placed on top of a piece of confetti that is in contact with the membrane on the insert. (c) Window (2 cm2) made in the egg shell of a 10-day-old chick embryo with organotypic culture transplanted. (d) After 10 days, implantation shell was opened and blood vessel points were observed; (e and f) note the wheel-spoke pattern of CAM blood vessels around the graft (undifferentiated and differentiated). Stimulation of angiogenesis in CAM by undifferentiated or differentiated hNSSCs. The expansion of new blood vessels was determined by counting branch points after 10 days of cell transplantation. Sprouting and branching vessels are prominent in the CAM transplanted by differentiated hNSSCs compared to the undifferentiated hNSSCs transplantation. (g) Quantification of new blood vessels formed by naive (undifferentiated) and differentiated hNSSCs compared to negative control (confetti without cell transplantation) (n = 5). ∗P < 0.05. (h, i) Paraffin sections of both undifferentiated and differentiated hNSSCs transplanted CAM were stained by haematoxylin and eosin stain to show the vascular density in CAM; blood vessels were counted again (blue colour points indicate vasculature) with Aperio's ImageScope software (Aperio Technologies, Vista, CA, USA) (Bar = 100 μm).
Mentions: hNSSCs were cultured on the CAM as previously detailed allowing the analysis of hNSSCs in an ex vivo culture (Figure 3) [12]. After Trypsinization, cell pellets were resuspended in 10 mL culture medium (control cells in routine culture medium, induced cells in endothelial induction medium). Cell count was performed to give a cell density of between 3 × 105 and 5 × 105/mL. One mL of cell suspension was added per falcon tube, centrifuged at 400 g for 10 minutes and incubated at 37°C, supplemented with 5% CO2. After 2 days, pellets formed were cultured for 2 more days on organotypic culture until cell attached to the confetti (Figures 3(a) and 3(b)) (hydrophilic PTFE (polytetrafluoroethylene) membranes (0.4 mm pore size); Millipore; UK).

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