Perinatal Stem Cells Overview Part 2

Perinatal Stem Cells (Part 2)

• Perinatal stem cells are derived from umbilical cord tissue, amniotic fluid, and amniotic-derived sources.

Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSCs)

• UC-MSCs are multipotent stem cells.
• They are primarily isolated from Wharton's jelly and the perivascular regions of the umbilical cord.
• They are easily and non-invasively isolated.

Properties of UC-MSCs

• Ease of Isolation: UC-MSCs are efficiently isolated from the umbilical cord, a readily available and non-invasive source.
• Reduced Ethical Concerns: Harvesting UC-MSCs does not harm the mother or baby and avoids ethical issues related to embryonic stem cells.
• Multipotency: UC-MSCs can differentiate into various cell types, including osteocytes (bone cells), chondrocytes (cartilage cells), and 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 makes them suitable for allogeneic transplantation.
• High Proliferative Capacity: UC-MSCs exhibit robust expansion potential, maintain viability and function for up to 30 passages, enabling large-scale production for therapeutic applications.
• Molecular Markers: Oct4 and Nanog are positive markers essential for maintaining stemness. CD45, CD34, and HLA-DR are negative markers, indicating they are non-hematopoietic and immunoprivileged.

Collection and Processing of UC-MSCs

• The umbilical cord (UC) is collected immediately after childbirth (vaginal or cesarean).
• A 10-20 cm segment is harvested, thoroughly washed with betadine and alcohol, and placed in a sterile container with a buffer solution.
• The buffer typically includes saline, heparin, albumin, antibiotics, and antifungals to keep the UC viable for processing at a specialized stem cell center for up to 48 hours.

Isolation Methods for UC-MSCs

• Enzymatic Digestion: Ringlets are treated with collagenase and/or hyaluronidase to digest the extracellular matrix and release MSCs.
• Direct Explant Culture: 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 of UC-MSCs

• Ringlets are incubated in a cryopreservation buffer (containing saline, sucrose, and DMSO) for 30 minutes at 4°C.
• The fragments are then frozen gradually in cryovials at -180°C and transferred to liquid nitrogen dewars for long-term storage.
• DMSO prevents the formation of intracellular ice crystals, preserving cell viability during freezing.

Amniotic Fluid-Derived Stem Cells (AFSCs)

• Amniotic fluid is derived from the amnion membrane, originating from the epiblast layer during early embryonic development.
• The epiblast is a component of the inner cell mass (ICM), giving rise to the ectoderm, mesoderm, and endoderm.

Properties of AFSCs

• Amniotic Fluid Function: Acts as a protective cushion for the fetus and has been historically used for determining fetal sex.
• Markers:
  • Positive Markers: Mesenchymal markers (CD73, CD90, CD105, CD166) and pluripotency markers (Oct4).
  • Negative Markers: Hematopoietic markers (CD45, CD34) and pluripotency markers (SSEA3, TRA1).
• Pluripotency Potential: Although not fully pluripotent like embryonic stem cells, AFSCs have higher differentiation potential than adult mesenchymal stem cells (MSCs).
• Multipotency: AFSCs can differentiate into cell types derived from all three germ layers (mesoderm, ectoderm, and endoderm).
• Immunomodulatory Effects: AFSCs secrete cytokines and growth factors to regulate immune responses, promote tissue repair, and reduce inflammation.
• 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, then cultured in appropriate growth media to isolate and expand AFSCs for therapeutic applications.

Amnion-Derived Stem Cells (ADSCs)

• Amnion-derived stem cells (ADSCs) are multipotent cells derived from the amnion membrane.
• They exhibit high proliferative capacity and valuable properties in regenerative medicine and immunotherapy.

Properties of ADSCs

• Two Types of Stem Cells: Amniotic epithelial cells (AECs) and mesenchymal stem cells (MSCs).
  • AECs have a cobblestone-like appearance in culture.
  • MSCs have a fibroblast-like morphology.
• Multipotency and Proliferative Capacity: Both AECs and MSCs are multipotent, capable of differentiating into various cell types and exhibiting higher expression of pluripotency markers (Oct4 and SSEA3).

Collection and Isolation of ADSCs

• ADSCs are collected from amniotic membranes of the placenta, typically post-delivery.
• The amniotic membrane is carefully washed, cut into small pieces, digested with enzymes (trypsin and collagenase).
• 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.

Applications

• Amniotic Epithelial Cells (AECs): Used in regenerative therapies for wound healing and corneal injuries.
• Mesenchymal Stem Cells (MSCs): Aid in regeneration and healing of bone, muscle, and cartilage tissues; used in immune modulation (treating autoimmune diseases and graft-versus-host disease).
• Chorion-derived MSCs: Similar in properties and characteristics to amnion-derived MSCs.