Reprogramming cells with synthetic proteins

Abstract

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

“…Synthetic biology is emerging as a provider of powerful tools for cellular reprogramming and stem cell biology with a focus on the CRISPR-Cas-based genome engineering. In addition, new opportunities arise to design artificial TFs with enhanced biological properties (reviewed in [53]). Our identification of critical elements underlying POU function and determining the unique properties of Oct4 can guide the generation of synthetic TFs that direct cellular identities as an alternative to CRISPR-Cas-based TF design.…”

Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping

“…Human fibroblasts are subjected to mRNA reprogramming [ 63 – 65 ] using a cocktail incorporating engineered, chimeric transcription factors to accelerate lineage conversion. The resulting iPSC colonies were stabilized and expanded under xeno-free conditions by Cellular Reprogramming, Inc. (Pasadena, CA).…”

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of α-synuclein inclusions

“…Fibroblasts from a male Iowa kindred patient (age 48) with severe early-onset parkinsonism were collected under Stanford protocol (IRB-15028) and previously described in 32,103 . Fibroblast cultures were subjected to mRNA-based reprogramming using engineered and chimeric transcription factors to facilitate lineage conversion [104][105][106] . Resulting induced pluripotent stem cell (iPSC) colonies were further expanded on rLaminin-521 (BioLamina, Sundbyberg, Sweden) in Nutristem XF media (Corning) for at least 3 passages prior to freezing; iPSCs were maintained on Matrigel in StemFlex Medium (Thermo Fisher Scientific).…”