Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons Skeletal Myocytes and Oligodendrocytes

摘要

class="head no_bottom_margin" id="sec1title">IntroductionDespite major efforts to develop robust protocols for scalable generation of human cell types from easily accessible and renewable sources, the differentiation of human pluripotent stem cells (hPSCs) into specific cell types often remains cumbersome, lengthy, and difficult to reproduce. Moreover, the recapitulation of developmental stages in vitro yields fetal cells that often do not reach full maturation (). More recently, forced expression of lineage-specific master regulators resulting in direct reprogramming of somatic cell types has provided an efficient alternative to directed differentiation (, , ). In particular, the direct conversion of hPSCs, termed forward programming (), combines the advantages of hPSC differentiation and direct cellular reprogramming, enabling scalable and rapid generation of human cell types ().Currently available forward programming protocols are largely based on lentiviral transduction of hPSCs, which results in variegated expression or complete silencing of transgenes (, ). Additional purification steps are usually necessary for enriching the desired cell type. Lentiviral approaches randomly insert transgenes into the genome bearing the risk of unwanted interference with the endogenous transcriptional program. Therefore, refinements to the current forward programming approaches are desirable.As the result of a systematic effort to optimize gene expression in hPSCs, we arrived at a robust hPSC forward programming platform by targeting all components of the Tet-ON system required for inducible expression of transcription factors into genomic safe harbor sites (GSHs) (). The Tet-ON system consists of two components: a constitutively expressed transcriptional activator protein responsive to doxycycline (dox) (reverse tetracycline transactivator [rtTA]), and an inducible promoter regulated by rtTA (Tet-responsive element) that drives expression of the transgene (). Previous GSH-targeting strategies of the Tet-ON system relied on introducing both elements into the AAVS1 GSH of hPSCs, either separately (), or together (using an all-in-one Tet-ON vector) (, ). Compared with these designs, we reasoned that targeting each of the two elements of the Tet-ON system into a different GSH would have several advantages: inducible overexpression based on dual GSH targeting would not be affected by promoter interference between the two transgenes (), while homozygous GSH targeting would maximize the number of safely targeted transgene copies. Moreover, the larger cargo capacity in each of the transgenes would permit increased flexibility for transgene design, thus allowing the insertion of large reprogramming cassettes.Here we show that an optimized approach based on dual GSH targeting of Tet-ON-controlled transgenes results in homogeneous, controllable, and extremely high expression of inducible transgenes in hPSCs. Application of the optimized overexpression platform enabled us to develop rapid and deterministic forward programming protocols for mature human cell types.