Perinatal Stem Cells Part 2 PDF

Umbilical cord tissue, Amnion Fluid & Amnion-Derived SCs Dr. Mohamed Elkhawanky Elkhawanky Associate Prof. of Hematopathology  Umbilical Cord-Derived Mesenchymal Stem Cells UC-MSCs are:  Multipotent stem cells.  Isolated mainl...

Umbilical cord tissue, Amnion Fluid & Amnion-Derived SCs Dr. Mohamed Elkhawanky Elkhawanky Associate Prof. of Hematopathology  Umbilical Cord-Derived Mesenchymal Stem Cells UC-MSCs are:  Multipotent stem cells.  Isolated mainly from Wharton’s jelly and the perivascular regions of the umbilical cord.  Ease of Isolation: ◦ UC-MSCs can be efficiently isolated from the umbilical cord, a readily available and non-invasive source.  Reduced Ethical Concerns: ◦ Harvesting UC-MSCs poses no harm to the mother or baby and avoids ethical controversies linked to embryonic stem cells.  Multipotency: ◦ UC-MSCs are capable of differentiating into a variety of cell types, including:  Osteocytes (bone cells)  Chondrocytes (cartilage cells)  Adipocytes (fat cells)  Immunomodulatory Effects: ◦ UC-MSCs modulate the immune system by releasing cytokines and growth factors that suppress inflammation and promote tissue repair. ◦ Their low immunogenicity, due to minimal expression of MHC-II and co-stimulatory molecules, makes them suitable for allogeneic transplantation.  High Proliferative Capacity: ◦ UC-MSCs exhibit robust expansion potential, maintaining viability and function for up to 30 passages, enabling large-scale production for therapeutic applications.  Molecular Markers: ◦ Positive Markers: Oct4 and Nanog, essential for maintaining stemness. ◦ Negative Markers: CD45, CD34, and HLA-DR, indicating they are non-hematopoietic and immunoprivileged.  Collection: ◦ The umbilical cord (UC) is collected immediately after childbirth (vaginal delivery or cesarean section). ◦ A segment measuring 10–20 cm is harvested, thoroughly washed with betadine and alcohol, and placed in a sterile container containing a buffer solution. ◦ This buffer typically includes saline, heparin, albumin, antibiotics, and antifungals, ensuring the UC remains viable for processing at a specialized stem cell (SC) center for up to 48 hours.  Preparation: ◦ The UC is washed again with alcohol and saline to ensure sterility. ◦ It is then sectioned into 5 mm ringlets using a scalpel or an automated cutting machine.  Isolation Methods for MSCs: ◦ Enzymatic Digestion: The ringlets are treated with collagenase and/or hyaluronidase to digest the extracellular matrix and release mesenchymal stem cells (MSCs). ◦ Direct Explant Culture: Alternatively, small fragments of Wharton’s jelly (WJ) are cultured directly in growth media. MSCs migrate from the tissue fragments and adhere to the culture surface, facilitating their expansion.  Cryopreservation: ◦ The ringlets are incubated in a cryopreservation buffer containing saline, sucrose, and dimethyl sulfoxide (DMSO) for 30 minutes at 4°C. ◦ The fragments are then frozen gradually in 4.5 cc cryovials at -180°C and transferred to liquid nitrogen dewars for long-term storage. ◦ Role of DMSO: DMSO prevents the formation of intracellular ice crystals, preserving cell viability during freezing.  Amniotic fluid is derived from the amnion membrane, which originates from the epiblast layer during early embryonic development.  The epiblast, a component of the inner cell mass (ICM), gives rise to the ectoderm, mesoderm, and endoderm, as well as extra- embryonic structures like the amnion.  Amniotic Fluid Function: ◦ Acts as a protective cushion for the fetus against mechanical injury. ◦ Historically used for determining fetal sex and resolving disputed paternity cases.  Markers: ◦ Positive Markers:  Mesenchymal markers: CD73, CD90, CD105, CD166.  Pluripotency markers: Oct4. ◦ Negative Markers:  Hematopoietic markers: CD45, CD34.  Pluripotency markers: SSEA3, TRA1. ◦ Pluripotency Potential:  While not fully pluripotent like embryonic stem cells (ESCs), AFSCs exhibit greater differentiation potential than adult mesenchymal stem cells (MSCs).  Multipotency: ◦ AFSCs can differentiate into cell types derived from all three germ layers:  Mesoderm: Osteocytes (bone), adipocytes (fat), and myocytes (muscle).  Ectoderm: Neurons and skin cells.  Endoderm: Hepatocytes (liver).  Immunomodulatory Effects: ◦ AFSCs secrete cytokines and growth factors to regulate immune responses, promote tissue repair, and reduce inflammation. ◦ Their low immunogenicity is attributed to minimal MHC-II expression, making them suitable for allogeneic applications.  Reduced Ethical Concerns: ◦ Amniotic fluid is safely collected during routine prenatal procedures or elective cesarean deliveries, causing no harm to the fetus or mother.  Processing and Isolation: ◦ Collected fluid is centrifuged to separate cells. ◦ Cells are cultured in appropriate growth media to isolate and expand AFSCs for therapeutic applications.  Amnion-derived stem cells (ADSCs) are multipotent cells. obtained from the amnion membrane.  They exhibit high proliferative capacity and possess properties that make them valuable in regenerative medicine and immunotherapy.  Two Types of Stem Cells: ◦ Amniotic Epithelial Cells (AECs): Characterized by a cobblestone-like appearance in culture. ◦ Mesenchymal Stem Cells (MSCs): Multipotent cells with fibroblast-like morphology.  Multipotency and Proliferative Capacity: ◦ Both AECs and MSCs are multipotent, capable of differentiating into various cell types. ◦ Exhibit higher expression of pluripotency markers, such as Oct4 and SSEA3.  Source: ◦ Collected from amniotic membranes of the placenta, typically post- delivery.  Isolation Process: ◦ Preparation:  The amniotic membrane is carefully washed and cut into small pieces. ◦ Enzymatic Digestion:  Digested with enzymes like trypsin and collagenase to release cells. ◦ Culture and Expansion:  Isolated cells are cultured in growth media containing essential growth factors. ◦ Once cells reach 70–80% confluency, they are passaged into new culture bottles for expansion to be used for therapeutic applications.  Amniotic Epithelial Cells (AECs): ◦ Wound Healing: Enhance tissue repair and reduce scarring. ◦ Corneal Injuries: Used in regenerative therapies for damaged corneal tissues.  Mesenchymal Stem Cells (MSCs): ◦ Bone, Muscle, and Cartilage Repair: Aid in regeneration and healing of skeletal and muscular tissues. ◦ Immune Modulation: Treat autoimmune diseases and Graft- versus-Host Disease (GvHD) by modulating the immune response.  Chorion-derived mesenchymal stem cells (MSCs) closely similar to amnion-derived MSCs in their properties and characteristics.