Fuglsanghartman5248

The pluripotency of human induced pluripotent stem cells (HiPSCs) cannot be tested strictly in a similar way as we can do for the mouse ones because of ethical restrictions. One common and initial approach to prove the pluripotency of an established human iPSC line is to demonstrate expression of a set of established surface and intracellular pluripotency markers. This chapter provides procedures of immunocytochemistry of the established HiPSC lines for a set of the signature intracellular pluripotency proteins, OCT4, SOX2, NANOG, and LIN28. We also describe cell phenotyping by flow cytometry for the five established human pluripotency surface markers, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and TRA2-49 (ALP). Numbers of ALP+ and TRA-1-60+ colonies are the most widely used parameters for evaluation of human iPSC reprogramming efficiency. Therefore, this chapter also provides detailed steps for substrate colorimetric reaction of the ALP activity, as well as the TRA-1-60 staining, of the iPSC colonies in the reprogramming population.Mouse embryonic fibroblasts (MEFs) can be used in co-culture to support generation of induced pluripotent stem cells (iPSCs) and the normal growth and proliferation of human pluripotent stem cells (hPSCs). Here, we describe the necessary steps to derive, expand, harvest, inactivate, plate, and use MEFs as feeders for iPSC generation and maintenance.The development of porcine expanded potential stem cells (pEPSCs) provides an invaluable tool for investigation of porcine stem cell pluripotency and opens a venue for research in biotechnology, agriculture, and regenerative medicine. Since the derivation of pEPSC from porcine pre-implantation embryos has been demanding in resource supply and technical challenges, it is more feasible and convenient for most laboratories to derive this new type of porcine stem cells by reprogramming somatic cells. In this chapter, we describe the detailed procedures for reprogramming porcine fetal fibroblast cells to EPSCiPSC with the eight reprogramming factors cloned on the piggyBac vectors followed by a selection for pluripotent cells independent of transgene expression using the EPSC media. This technique allows the generation of pEPSCs for stem cell research, genome editing, biotechnology, and agriculture.CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components. This chapter also provides procedure to assemble guide RNA cassettes and generation of multiplexed guide plasmids for readers who want to design their own guide RNAs.Human-induced pluripotent stem cells (iPSCs) are showing great promise for both disease modeling and regenerative medicine. The choice of reprogramming methods have a significant effect on the outcomes of the experiments. Standard methods, such as Sendai viruses, episomes, and the base-modified mRNA have limitations. Here, I describe a method to reprogram human fibroblasts using a cocktail of mRNAs without any base modification that increases reprogramming efficiency, reduces the RNA-associated toxicity, and yields iPSCs ready for expansion and characterization in as short as 10-14 days.The discovery of induced pluripotent stem cells (iPSCs) allows for establishment of human embryonic stem-like cells from various adult human somatic cells (e.g., fibroblasts), without the need for destruction of human embryos. This provides an unprecedented opportunity where patient-specific iPSCs can be subsequently differentiated to many cell types, e.g., cardiac cells and neurons, so that we can use these iPSC-derived cells to study patient-specific disease mechanisms and conduct drug testing and screening. Critically, these cells have unlimited therapeutic potentials, and there are many ongoing clinical trials to investigate the regenerative potentials of these iPSC-derivatives in humans. However, the traditional iPSC reprogramming methods have problem of insertional mutagenesis because of use of the integrating viral vectors. While a number of advances have been made to mitigate this issue, including the use of chemicals, excisable and non-integrating vectors, and use of the modified mRNA, safety remains a concern. Both integrating and non-integrating methods also suffer from many other limitations including low efficiency, variability, and tumorigenicity. The non-integrating mRNA reprogramming is of high efficiency, but it is sensitive to reagents and need approaches to reduce the immunogenic reaction. An alternative non-integrating and safer way of generating iPSCs is via direct delivery of recombinant cell-penetrating reprogramming proteins into the cells to be reprogrammed, but reprogramming efficiency of the protein-based approach is extremely low compared to the conventional virus-based nuclear reprogramming. Herein, we describe detailed steps for efficient generation of human iPSCs by protein-based reprogramming in combination with stimulation of the Toll-like receptor 3 (TLR3) innate immune pathway.Human-induced pluripotent stem cells (iPSCs) can be generated from patient-specific somatic cells by forced expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Sustained expression of the transgenes during reprogramming is crucial for the successful derivation of iPSCs. Integrating retroviruses have been used to achieve the required prolonged expression; however, issues of undesirable transgene expression in the iPSC-derived cell types post reprogramming can occur. selleck Alternative non-integrating approaches to reprogram somatic cells into pluripotency have been established. Here, we describe a detailed method for generating human iPSCs from fibroblasts and peripheral blood mononuclear cells (PBMCs) using the non-integrating episomal plasmids. The delivery of the episomal plasmids into the somatic cells is achieved using a nucleofection technique, and reprogramming is performed in chemically defined media. This process takes approximately 30 days to establish the iPSC colonies. We also describe a method for growing iPSCs on vitronectin as well as procedures for the long-term expansion of iPSCs on human fibroblast feeder cells.