Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimentional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.
Non-alcoholic fatty liver disease (NAFLD) affects 30 to 40% of adults and 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to be a leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes for NASH modeling applications and identification of potential drug targets. We developed a novel defined in vitro differentiation process to generate cryopreservable hepatocytes using an iPSC panel of NASH donors and apparently healthy normal (AHN) controls. iPSC-derived hepatocytes displayed stage specific phenotypic markers, hepatocyte morphology, with bile canaliculi. Importantly, both fresh and cryopreserved Definitive Endoderm and Hepatoblasts successfully differentiated to pure and functional hepatocytes with increased CYP3A4 activity in response to rifampicin and lipid accumulation upon fatty acid (FA) treatment. End stage hepatocytes integrated into three dimensional liver organoids and demonstrated increased levels of albumin secretion compared to aggregates consisting of hepatocytes alone. End stage hepatocytes derived from NASH donors demonstrated spontaneous lipidosis without fatty acid supplementation, recapitulating a feature of NASH hepatocytes in vivo. Cryopreserved hepatocytes generated by this protocol across multiple donors will provide a critical cell source to facilitate the fundamental understanding of NAFLD/NASH biology and potential high throughput screening applications for preclinical evaluation of therapeutic targets.
Macrophages are innate immune cells that play critical roles in tissue homeostasis, inflammation, and immune oncology. Macrophages differentiated from human induced pluripotent stem cells (iPSCs) overcome many limitations of using peripheral blood derived macrophages. The ability to scale up and cryopreserve a large amount of end stage macrophages from single clonal iPSCs from normal and disease specific donors offers a unique opportunity for genomic analysis and drug screening. The present study describes the step wise generation and characterization of macrophages from iPSCs using a defined serum free method amenable to scale up to generate a large batch of pure end stage cryopreservable macrophages expressing CD68, CD33, CD11c, CD11b, CD1a, HLA-DR, CD86, CD64, CD80, CD206, CD169, CD47, HLA-ABC, and CX3CR. The end stage macrophages pre and post cryopreservation retain purity, morphology, responsiveness to stimuli and display robust phagocytic function coming right out of cryopreservation. The same differentiation process was used to generate end stage macrophages from isogenic iPSCs engineered to mimic mutations associated with Parkinson’s disease (SNCA A53T), neuronal ceroid lipofuscinosis (GRN2/GRN R493X), and Rett syndrome (MECP2-Knockout). End stage macrophages from isogenic engineered clones displayed differential macrophage-specific purity markers, phagocytic function, and response to specific stimuli. Thus, generating a panel of functional, physiologically relevant iPSC-derived macrophages can potentially facilitate the understanding of neural inflammatory responses associated with neurodegeneration.
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