Amniotic Fluid and Placental Membranes
Amniotic Fluid and Placental Membranes
Mesenchymal stromal cells (MSCs) were initially identified in the bone marrow of adult subjects. MSCs contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and stroma. MSCs have also been derived from other nonmarrow tissues such as adipose tissue, adult muscle, dental pulp, and gestational tissue. Within the last decade, several groups published relatively simple protocols for isolating a nonspecific population of cells with mesenchymal-like characteristics from the amniotic fluid and placenta. MSCs have the ability to differentiate into osteocytes, chondrocytes, and adipocytes in culture and possess immunomodulatory properties. These criteria are commonly used for defining bone-marrow MSCs:
Cells fitting these criteria have been isolated from the amniotic fluid, amnion, and chorion membranes as well as from the amniotic fluid. Steigman and Fauza described a method of isolating MSCs from the placenta where the fetal placental specimen is mechanically separated from the maternal decidua, either by blunt dissection or during the process of chorionic villus sampling. The remaining mesenchymal core is minced and digested with a collagenase/dispase mixture, filtered and plated on cover slips. Placental MSCs share with adult MSCs a spindle-shaped fibroblast appearance, the ability to adhere to plastic and to expand ex vivo, which are essential properties for isolation. However, placental MSCs expand quicker in vitro than adult MSCs and appear to be both less immunogenic and more immunosuppressive than their adult counterpart. These properties make placental MSCs attractive as a potential clinical therapy for inflammatory diseases.
A multipotent epithelial cell population has also been isolated from the amnion membrane. Human amnion epithelial cells (hAECs) have attracted attention as a potential source of cells for regenerative therapies, with reports that epithelial cells derived from human term amnion possess multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. With a view to future clinical applications, our group demonstrated that hAECs can be collected, isolated, and stored in a manner suitable for clinical therapies. Term human amnion membrane is manually separated from the chorion membrane and washed in a saline solution to remove blood. Epithelial cells are isolated from the amnion by enzymatic digestion in TrypZean, an animal product-free recombinant trypsin. Approximately 120 million viable epithelial cells can be isolated from a single amnion membrane, and these cells can be maintained in serum-free culture conditions and display a normal karyotype and cell cycle distribution following culture while maintaining long telomere lengths. Although hAECs lack the proliferative capacity of placental MSCs, they are highly multipotent, differentiating into cells representing all three germ layers, specifically osteocytes, adipocytes, neurons, lung epithelial cells, cardiomyocytes, myocytes, hepatocytes, and pancreatic cells. The immunosuppressive properties of hAECs have been well characterized both in vitro and in vivo.
It has been proposed that soluble factors produced by hAECs have anti-inflammatory effects and act to inhibit both the innate and adaptive immune systems. Thus hAECs may have important applications as a clinical cell therapy for a multitude of congenital and adult disorders.
AFSCs can be obtained from a small amount of fluid during amniocentesis at the second trimester. There is also the potential to collect amniotic fluid at term from routine cesarean deliveries. Kaviani and coworkers reported that just 2 mL of amniotic fluid contains up to 20,000 cells, 80% of which are viable. A highly multipotent subpopulation of AFSCs present in the amniotic fluid and placenta can be isolated through positive selection for cells expressing the membrane receptor c-kit (CD117). C-kit is expressed on a variety of stem cells including embryonic stem cells (ESCs), primordial germ cells, and many somatic stem cells. Approximately 1% of cells present in amniotic fluid have been shown to be c-kit positive. Progenitor cells maintain a round shape for 1-week after isolation when cultured in nontreated culture dishes. In this state, they demonstrate low proliferative capability. After the first week the cells begin to adhere to the plate and change their morphology, becoming elongated and proliferating rapidly. AFSCs show a high self-renewal capacity and maintain a normal karyotype at late passages and display normal G1 and G2 cell cycle checkpoints. They also conserve a long telomere length in late passages due to continued telomerase activity. Like ESCs, AFSCs form embryoid bodies in vitro that stain positive for markers of all three germ layers. However, unlike embryonic stem cells, when implanted into immunodeficient mice in vivo, AFSCs do not form teratomas, an essential safety characteristic for a potential cell therapy. AFSC have a high clonal capacity demonstrated using a technique involving retrovirally tagged cells. In this assay, a tagged single cell gave rise to a population that differentiated along six distinct lineages from all three germ layers: adipogenic, osteogenic, myogenic, endothelial, neurogenic, and hepatic.
Cells Derived From Amniotic Fluid and Placenta
Placental Mesenchymal Cells
Mesenchymal stromal cells (MSCs) were initially identified in the bone marrow of adult subjects. MSCs contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and stroma. MSCs have also been derived from other nonmarrow tissues such as adipose tissue, adult muscle, dental pulp, and gestational tissue. Within the last decade, several groups published relatively simple protocols for isolating a nonspecific population of cells with mesenchymal-like characteristics from the amniotic fluid and placenta. MSCs have the ability to differentiate into osteocytes, chondrocytes, and adipocytes in culture and possess immunomodulatory properties. These criteria are commonly used for defining bone-marrow MSCs:
Adherence to plastic
Formation of fibroblast colony-forming units
Differentiation potential toward one or more mesodermal lineages
Express CD90, CD73, and CD105
Do not express CD45, CD34, CD14, or HLA-DR
Cells fitting these criteria have been isolated from the amniotic fluid, amnion, and chorion membranes as well as from the amniotic fluid. Steigman and Fauza described a method of isolating MSCs from the placenta where the fetal placental specimen is mechanically separated from the maternal decidua, either by blunt dissection or during the process of chorionic villus sampling. The remaining mesenchymal core is minced and digested with a collagenase/dispase mixture, filtered and plated on cover slips. Placental MSCs share with adult MSCs a spindle-shaped fibroblast appearance, the ability to adhere to plastic and to expand ex vivo, which are essential properties for isolation. However, placental MSCs expand quicker in vitro than adult MSCs and appear to be both less immunogenic and more immunosuppressive than their adult counterpart. These properties make placental MSCs attractive as a potential clinical therapy for inflammatory diseases.
Amnion Epithelial Cells
A multipotent epithelial cell population has also been isolated from the amnion membrane. Human amnion epithelial cells (hAECs) have attracted attention as a potential source of cells for regenerative therapies, with reports that epithelial cells derived from human term amnion possess multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. With a view to future clinical applications, our group demonstrated that hAECs can be collected, isolated, and stored in a manner suitable for clinical therapies. Term human amnion membrane is manually separated from the chorion membrane and washed in a saline solution to remove blood. Epithelial cells are isolated from the amnion by enzymatic digestion in TrypZean, an animal product-free recombinant trypsin. Approximately 120 million viable epithelial cells can be isolated from a single amnion membrane, and these cells can be maintained in serum-free culture conditions and display a normal karyotype and cell cycle distribution following culture while maintaining long telomere lengths. Although hAECs lack the proliferative capacity of placental MSCs, they are highly multipotent, differentiating into cells representing all three germ layers, specifically osteocytes, adipocytes, neurons, lung epithelial cells, cardiomyocytes, myocytes, hepatocytes, and pancreatic cells. The immunosuppressive properties of hAECs have been well characterized both in vitro and in vivo.
It has been proposed that soluble factors produced by hAECs have anti-inflammatory effects and act to inhibit both the innate and adaptive immune systems. Thus hAECs may have important applications as a clinical cell therapy for a multitude of congenital and adult disorders.
Amniotic Fluid Stem Cells
AFSCs can be obtained from a small amount of fluid during amniocentesis at the second trimester. There is also the potential to collect amniotic fluid at term from routine cesarean deliveries. Kaviani and coworkers reported that just 2 mL of amniotic fluid contains up to 20,000 cells, 80% of which are viable. A highly multipotent subpopulation of AFSCs present in the amniotic fluid and placenta can be isolated through positive selection for cells expressing the membrane receptor c-kit (CD117). C-kit is expressed on a variety of stem cells including embryonic stem cells (ESCs), primordial germ cells, and many somatic stem cells. Approximately 1% of cells present in amniotic fluid have been shown to be c-kit positive. Progenitor cells maintain a round shape for 1-week after isolation when cultured in nontreated culture dishes. In this state, they demonstrate low proliferative capability. After the first week the cells begin to adhere to the plate and change their morphology, becoming elongated and proliferating rapidly. AFSCs show a high self-renewal capacity and maintain a normal karyotype at late passages and display normal G1 and G2 cell cycle checkpoints. They also conserve a long telomere length in late passages due to continued telomerase activity. Like ESCs, AFSCs form embryoid bodies in vitro that stain positive for markers of all three germ layers. However, unlike embryonic stem cells, when implanted into immunodeficient mice in vivo, AFSCs do not form teratomas, an essential safety characteristic for a potential cell therapy. AFSC have a high clonal capacity demonstrated using a technique involving retrovirally tagged cells. In this assay, a tagged single cell gave rise to a population that differentiated along six distinct lineages from all three germ layers: adipogenic, osteogenic, myogenic, endothelial, neurogenic, and hepatic.
Source...