Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.
Male ; Mice ; Animals ; Cellular Reprogramming/genetics* ; Tetraploidy ; Pluripotent Stem Cells/metabolism* ; Induced Pluripotent Stem Cells/metabolism* ; DNA Methylation ; Spermatogonia/metabolism* ; Germ Cells/metabolism*

Animals ; Cellular Reprogramming ; genetics ; DNA Transposable Elements ; genetics ; Genetic Vectors ; genetics ; Genome ; genetics ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Mutagenesis

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.
Animals ; Apoptosis ; Base Sequence ; CLOCK Proteins ; genetics ; metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; Cellular Reprogramming ; Circadian Clocks ; genetics ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Hepatocyte Nuclear Factor 3-beta ; genetics ; metabolism ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; SOXB1 Transcription Factors ; genetics ; metabolism

OBJECTIVETo observe the histological features of tumor-bearing tissues formed by human fibroblasts after reprograming by spermatogonial stem cell self-renewal key regulating gene Piwil2 (Piwil2-iCSC).

METHODSPiwil2-iCSC tumor spheroids-like colonies were selected for tumor formation assay in four nude mice. Pathological features of Piwil2-iCSC tumors were observed by histology. Stem cell markers and common triploblastic markers were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) assay and immunohistochemistry. Germ cell tumor markers were detected by immunohistochemical examination.

RESULTSTwo weeks after inoculation, subcutaneous tumors were formed in all the four nude mice with a tumor formation rate of 100%. In the Piwil2-iCSC tumor tissues, Piwil2-GFP(+) cells showed high-density nuclear expression and were widely observed in DAPI-stained sections. Numerous mitotic figure of the neoplastic cells were seen (>10 cells/field of vision under high magnification) in HE-stained sections. Enlarged abnormal cell nuclei were observed. RT-PCR assay showed that Piwil2-iCSC tumors still expressed Piwil2 and some self-renewal and pluripotent markers of stem cells and some markers of triploblastic differentiation. Immunohistochemical staining showed that the tumors expressed stem cell markers, triploblastic markers and germ cell tumor markers AFP and HCG.

CONCLUSIONSPiwil2-iCSC tumors are probably undifferentiated embryonic small cell carcinoma, most likely to be immature teratoma, mixed with yolk sac tumor and choriocarcinoma components. It can be used as a useful model for the research of origin or genesis mechanism of cancer stem cells and the treatment of relevant tumors.

Adult Stem Cells ; Animals ; Argonaute Proteins ; genetics ; Cellular Reprogramming Techniques ; Choriocarcinoma ; pathology ; Endodermal Sinus Tumor ; pathology ; Fibroblasts ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Mice ; Mice, Nude ; Neoplasms, Germ Cell and Embryonal ; chemistry ; genetics ; pathology ; Neoplastic Stem Cells ; chemistry ; pathology ; Real-Time Polymerase Chain Reaction ; Spheroids, Cellular ; Teratoma ; pathology ; Time Factors

Cell fate conversion is considered as the changing of one type of cells to another type including somatic cell reprogramming (de-differentiation), differentiation, and trans-differentiation. Epithelial and mesenchymal cells are two major types of cells and the transitions between these two cell states as epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) have been observed during multiple cell fate conversions including embryonic development, tumor progression and somatic cell reprogramming. In addition, MET and sequential EMT-MET during the generation of induced pluripotent stem cells (iPSC) from fibroblasts have been reported recently. Such observation is consistent with multiple rounds of sequential EMT-MET during embryonic development which could be considered as a reversed process of reprogramming at least partially. Therefore in current review, we briefly discussed the potential roles played by EMT, MET, or even sequential EMT-MET during different kinds of cell fate conversions. We also provided some preliminary hypotheses on the mechanisms that connect cell state transitions and cell fate conversions based on results collected from cell cycle, epigenetic regulation, and stemness acquisition.
Animals ; Cell Differentiation ; Cell Lineage ; Cellular Reprogramming ; Epigenesis, Genetic ; genetics ; Epithelial-Mesenchymal Transition ; Humans ; Induced Pluripotent Stem Cells ; cytology

BACKGROUNDSince an effective method for generating induced pluripotent stem cells (iPSCs) from human neural stem cells (hNSCs) can offer us a promising tool for studying brain diseases, here we reported direct reprogramming of adult hNSCs into iPSCs by retroviral transduction of four defined factors.

METHODSNSCs were successfully isolated and cultured from the hippocampus tissue of epilepsy patients. When combined with four factors (OCT3/4, SOX2, KLF4, and c-MYC), iPSCs colonies were successfully obtained.

RESULTSMorphological characterization and specific genetic expression confirmed that these hNSCs-derived iPSCs showed embryonic stem cells-like properties, which include the ability to differentiate into all three germ layers both in vitro and in vivo.

CONCLUSIONOur method would be useful for generating human iPSCs from NSCs and provide an important tool for studying neurological diseases.

Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Cellular Reprogramming ; genetics ; physiology ; Humans ; Immunohistochemistry ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Kruppel-Like Transcription Factors ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Octamer Transcription Factor-3 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SOXB1 Transcription Factors ; metabolism

The somatic epigenome can be reprogrammed to a pluripotent state by a combination of transcription factors. Altering cell fate involves transcription factors cooperation, epigenetic reconfiguration, such as DNA methylation and histone modification, posttranscriptional regulation by microRNAs, and so on. Nevertheless, such reprogramming is inefficient. Evidence suggests that during the early stage of reprogramming, the process is stochastic, but by the late stage, it is deterministic. In addition to conventional reprogramming methods, dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve induced pluripotent stem cell (iPSC) reprogramming. Indeed, iPS cells have been created recently using chemical compounds only. iPSCs are thought to display subtle genetic and epigenetic variability; this variability is not random, but occurs at hotspots across the genome. Here we discuss the progress and current perspectives in the field. Research into the reprogramming process today will pave the way for great advances in regenerative medicine in the future.
Animals ; Cell Differentiation ; Cellular Reprogramming ; MicroRNAs ; genetics ; Models, Biological ; Pluripotent Stem Cells ; cytology ; metabolism ; Stochastic Processes

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specifi c iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.
Cell Differentiation ; Cell Lineage ; Cell Transdifferentiation ; Cellular Reprogramming ; drug effects ; Embryonic Stem Cells ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; transplantation ; Neural Stem Cells ; cytology ; transplantation ; Neurodegenerative Diseases ; therapy ; Regenerative Medicine ; Transcription Factors ; genetics ; metabolism

Ascorbic Acid ; chemistry ; pharmacology ; Cellular Reprogramming ; Genetic Vectors ; genetics ; metabolism ; Histone Deacetylases ; genetics ; metabolism ; Humans ; Induced Pluripotent Stem Cells ; cytology ; drug effects ; Protein Kinase Inhibitors ; chemistry ; pharmacology ; RNA, Small Interfering ; metabolism

Nuclear reprogramming is described as a molecular switch, triggered by the conversion of one cell type to another. Several key experiments in the past century have provided insight into the field of nuclear reprogramming. Previously deemed impossible, this research area is now brimming with new findings and developments. In this review, we aim to give a historical perspective on how the notion of nuclear reprogramming was established, describing main experiments that were performed, including (1) somatic cell nuclear transfer, (2) exposure to cell extracts and cell fusion, and (3) transcription factor induced lineage switch. Ultimately, we focus on (4) transcription factor induced pluripotency, as initiated by a landmark discovery in 2006, where the process of converting somatic cells to a pluripotent state was narrowed down to four transcription factors. The conception that somatic cells possess the capacity to revert to an immature status brings about huge clinical implications including personalized therapy, drug screening and disease modeling. Although this technology has potential to revolutionize the medical field, it is still impeded by technical and biological obstacles. This review describes the effervescent changes in this field, addresses bottlenecks hindering its advancement and in conclusion, applies the latest findings to overcome these issues.
Animals ; Cell Fusion ; Cell Lineage ; genetics ; Cellular Reprogramming ; genetics ; Humans ; Nuclear Transfer Techniques ; Pluripotent Stem Cells ; cytology ; metabolism ; Transcription Factors ; metabolism