Differentiation of human induced pluripotent stem cells into functional lung alveolar epithelial cells in 3D dynamic culture.

Introduction: Understanding lung epithelium cell development from human induced pluripotent stem cells (IPSCs) in vitro can lead to an individualized model for lung engineering, therapy, and drug testing. Method: We developed a protocol to produce lung mature type I pneumocytes using encapsulation of human IPSCs in 1.1% (w/v) alginate solution within a rotating wall bioreactor system in only 20 days without using feeder cells. The aim was to reduce exposure to animal products and laborious interventions in the future. Results: The three-dimensional (3D) bioprocess allowed cell derivation into endoderm, and subsequently into type II alveolar epithelial cells within a very short period. Cells successfully expressed surfactant proteins C and B associated with type II alveolar epithelial cells, and the key structure of lamellar bodies and microvilli was shown by transmission electron microscopy. The survival rate was the highest under dynamic conditions, which reveal the possibility of adapting this integration for large-scale cell production of alveolar epithelial cells from human IPSCs. Discussion: We were able to develop a strategy for the culture and differentiation of human IPSCs into alveolar type II cells using an in vitro system that mimics the in vivo environment. Hydrogel beads would offer a suitable matrix for 3D cultures and that the high-aspect-ratio vessel bioreactor can be used to increase the differentiation of human IPSCs relative to the results obtained with traditional monolayer cultures.

Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells.

Introduction: High-efficacy single-cell cloning of human-induced pluripotent cells (IPSCs) remains a major challenge. The development of a culture method that supports single-cell passaging while maintaining reproducibility, homogeneity, scalability, and cell expansion to clinically relevant numbers is necessary for clinical application. Methods: To address this issue, we combined the use of the rho-associated protein kinase (ROCK) inhibitor Y-27632 and hypoxic conditions in culture to produce a novel, efficient single-cell culture method for human IPSCs and embryonic stem cells. Results: Through immunocytochemistry, alkaline phosphatase assays, and flow cytometry, we demonstrated that our method enabled high single-cell proliferation while maintaining self-renewal and pluripotency abilities. Discussion: We showed the beneficial effect of the interaction between hypoxia and ROCK inhibition in regulating cell proliferation, pluripotency, and single-cell survival of pluripotent cells.

CD166(pos) subpopulation from differentiated human ES and iPS cells support repair of acute lung injury.

Previous efforts to derive lung progenitor cells from human embryonic stem (hES) cells using embryoid body formation or stromal feeder cocultures had been limited by low efficiencies. Here, we report a step-wise differentiation method to drive both hES and induced pluripotent stem (iPS) cells toward the lung lineage. Our data demonstrated a 30% efficiency in generating lung epithelial cells (LECs) that expresses various distal lung markers. Further enrichment of lung progenitor cells using a stem cell marker, CD166 before transplantation into bleomycin-injured NOD/SCID mice resulted in enhanced survivability of mice and improved lung pulmonary functions. Immunohistochemistry of lung sections from surviving mice further confirmed the specific engraftment of transplanted cells in the damaged lung. These cells were shown to express surfactant protein C, a specific marker for distal lung progenitor in the alveoli. Our study has therefore demonstrated the proof-of-concept of using iPS cells for the repair of acute lung injury, demonstrating the potential usefulness of using patient's own iPS cells to prevent immune rejection which arise from allogenic transplantation.

Stem cells bioprocessing: an important milestone to move regenerative medicine research into the clinical arena.

Regenerative Medicine is a new, multidisciplinary field that combines expertise in biology, chemistry, engineering, materials, and medicine, to find solutions to some of the most challenging medical problems faced by humankind. Regenerative Medicine has the potential to impact the whole spectrum of health care, such as heart disease, emphysema, and diabetes. Regenerative Medicine employs various combinations of specially grown cells, tissues, and laboratory-made compounds to replace or amplify the body's natural healing process. The impact of Regenerative Medicine to the health care industry is likely to be comparable with that of antibiotics, vaccines and lately, monoclonal antibodies have had in clinical care. Regenerative Medicine is growing and maturing steadily; however, many challenges lie ahead. These include best cell source, most appropriate biomaterials, and reliable ways of expanding the cells and growing them in a three-dimensional environment (stem cell bioprocessing). This concise review deals with current achievements in the field, challenges that lie ahead and potential ways of having robust and reliable "off the shelf" cellular products.