ABSTRACT
Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.
Subject(s)
Human Embryonic Stem Cells/metabolism , Transcription Factors/metabolism , Cell Differentiation/physiology , Cell Line , Human Embryonic Stem Cells/cytology , Humans , Single-Cell Analysis/methodsABSTRACT
The derivation of kidney tissues from human pluripotent stem cells (hPSCs) and its application for replacement therapy in end-stage renal disease have been widely discussed. Here we report that consecutive transfections of two sets of synthetic mRNAs encoding transcription factors can induce rapid and efficient differentiation of hPSCs into kidney tissues, termed induced nephron-like organoids (iNephLOs). The first set - FIGLA, PITX2, ASCL1 and TFAP2C, differentiated hPSCs into SIX2+SALL1+ nephron progenitor cells with 92% efficiency within 2 days. Subsequently, the second set - HNF1A, GATA3, GATA1 and EMX2, differentiated these cells into PAX8+LHX1+ pretubular aggregates in another 2 days. Further culture in both 2-dimensional and 3-dimensional conditions produced iNephLOs containing cells characterized as podocytes, proximal tubules, and distal tubules in an additional 10 days. Global gene expression profiles showed similarities between iNephLOs and the human adult kidney, suggesting possible uses of iNephLOs as in vitro models for kidneys.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kidney/cytology , Kidney/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , RNA, Messenger/genetics , Transcription Factors/genetics , Biomarkers , Cell Culture Techniques , Cell Differentiation/genetics , Cell Lineage/genetics , Fluorescent Antibody Technique , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Immunophenotyping , Models, Biological , Nephrons , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolismABSTRACT
Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.
