Abstract
Experiments with somatic cell nuclear transfer, inter-cellular hybrid formation_ENREF_3, and ectopic expression of transcription factors have clearly demonstrated that cell fate can be dramatically altered by changing the epigenetic state of cell nuclei. Here we demonstrate, using chemical fusion, direct reprogramming of the genome of human embryonic fibroblasts (HEF) into the state of human fetal liver hFL CD34+ (hFL) hematopoietic progenitors capable of proliferating and differentiating into multiple hematopoietic lineages. We show that hybrid cells retain their ploidy and can differentiate into several hematopoietic lineages. Hybrid cells follow transcription program of differentiating hFL cells as shown by genome-wide transcription profiling. Using whole-genome single nucleotide polymorphism (SNP) profiling of both donor genomes we demonstrate reprogramming of HEF genome into the state of hFL hematopoietic progenitors. Our results prove that it is possible to convert the fetal somatic cell genome into the state of fetal hematopoietic progenitors by fusion. This suggests a possibility of direct reprogramming of human somatic cells into tissue specific progenitors/stem cells without going all the way back to the embryonic state. Direct reprogramming of terminally differentiated cells into the tissue specific progenitors will likely prove useful for the development of novel cell therapies.
Highlights
Somatic cells have been reprogrammed into the embryonic state [1,2,3,4,5,6], as well as into several types of terminally differentiated cells, including myoblasts [7], macrophages [8], beta-cells [9], and neurons [10]
Cells were synchronized in mitosis because we hypothesized that the dispersion of transcription factors through the cytoplasm in metaphase as a result of the nuclear membrane break down might contribute to a temporary relaxation of the transcriptional control that defines cell identity and may favor reprogramming [16]
human embryonic fibroblasts (HEF) were transduced with retroviral vectors expressing antip53 short hairpin RNA (shRNA) and a puromycin resistance marker and selected for puromycin resistance. human fetal liver CD34+ (hFL) cells were transduced with a lentivirus expressing GFP and anti-p53 shRNA and selected using fluorescence activated cell sorting (FACS) (Fig. 1A, B, C; see Materials and Methods)
Summary
Somatic cells have been reprogrammed into the embryonic state [1,2,3,4,5,6], as well as into several types of terminally differentiated cells, including myoblasts [7], macrophages [8], beta-cells [9], and neurons [10]. The conversion of the somatic cell genome into a state of tissue-specific stem cells/progenitors has not been demonstrated before now. Embryonic stem (ES) cells can differentiate into many cell types but can remain phenotypically and transcriptionally stable in vitro in the appropriate culture conditions. Most terminally differentiated cells are phenotypically and transcriptionally stable both in vitro and in vivo. Hematopoietic progenitors are uni-, bi-, or multi-potent since they can differentiate into mature blood cells types [11], but have a limited self-renewal capacity and are transcriptionally unstable in vivo. We demonstrate the reprogramming through cell fusion of the genome of human embryonic fibroblasts (HEF) into a transcriptionally unstable state of human fetal liver CD34+ (hFL) hematopoietic progenitors capable of proliferating and differentiating into multiple hematopoietic lineages
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