Attenuating iPSC reprogramming stress with dominant-negative BET peptides.

Generation of induced pluripotent stem cells (iPSCs) is inefficient and stochastic. The underlying causes for these deficiencies are elusive. Here, we showed that the reprogramming factors (OCT4, SOX2, and KLF4, collectively OSK) elicit dramatic reprogramming stress even without the pro-oncogene MYC including massive transcriptional turbulence, massive and random deregulation of stress-response genes, cell cycle impairment, downregulation of mitotic genes, illegitimate reprogramming, and cytotoxicity. The conserved dominant-negative (DN) peptides of the three ubiquitous human bromodomain and extraterminal (BET) proteins enhanced iPSC reprogramming and mitigated all the reprogramming stresses mentioned above. The concept of reprogramming stress developed here affords an alternative avenue to understanding and improving iPSC reprogramming. These DN BET fragments target a similar set of the genes as the BET chemical inhibitors do, indicating a distinct approach to targeting BET proteins.

Reprogramming Human Fibroblasts to Induced Pluripotent Stem Cells Using the GFP-Marked Lentiviral Vectors in the Chemically Defined Medium.

Much investigation is needed to understand the underlying molecular mechanisms of iPSC reprogramming and to improve this technology. Lentivirus-mediated iPSC reprogramming remains the most effective method to study human pluripotency reprogramming. iPSC production is more efficient and consistent in the chemically defined medium. Fibroblasts are the most common starting cells for iPSC generation. Here, we provide a detailed protocol for iPSC generation from human fibroblasts using the GFP-expressing lentiviral vectors in the chemically defined medium.

Evaluating Reprogramming Efficiency and Pluripotency of the Established Human iPSCS by Pluripotency Markers.

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.

Wnt/ß-Catenin/TCF Pathway Is a Phase-Dependent Promoter of Colony Formation and Mesendodermal Differentiation During Human Somatic Cell Reprogramming.

Somatic cell reprogramming is a biphasic phenomenon that goes through a mesenchymal-to-epithelial transition, called initiation phase, followed by a maturation phase wherein reprogramming cells acquire pluripotency. Here, we show that these phases display a differential response to Wnt signaling activation. Wnt signaling increases colony formation by promoting cellular epithelialization during the initiation phase in a TCF7-dependent manner. However, during maturation phase, it is also responsible for inducing mesendodermal differentiation, which is negatively regulated by TCF7L1. Thus, Wnt signaling inhibition or TCF7L1 overexpression downregulates mesendodermal gene expression without perturbing pluripotency. Together, our results demonstrate that a phase-specific modulation of Wnt signaling leads to an improved reprogramming efficiency in terms of colony output and pluripotency acquisition. This work provides new insights into the cell context-dependent roles of Wnt signaling during human somatic cell reprogramming. Stem Cells 2018;36:683-695.