Anne Mari Håkelien

Reprogramming fibroblasts to express T-cell functions using cell extracts

We demonstrate here the functional reprogramming of a somatic cell using a nuclear and cytoplasmic extract derived from another somatic cell type. Reprogramming of 293T fibroblasts in an extract from primary human T cells or from a transformed T-cell line is evidenced by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex, histone acetylation, and activation of lymphoid cell–specific genes. Reprogrammed cells express T cell–specific receptors and assemble the interleukin-2 receptor in response to T cell receptor–CD3 (TCR–CD3) complex stimulation. Reprogrammed primary skin fibroblasts also express T cell–specific antigens. After exposure to a neuronal precursor extract, 293T fibroblasts express a neurofilament protein and extend neurite-like outgrowths. In vitro reprogramming of differentiated somatic cells creates possibilities for producing isogenic replacement cells for therapeutic applications.

Reprogrammed gene expression in a somatic cell-free extract

We have developed a somatic cell‐free system that remodels chromatin and activates gene expression in heterologous differentiated nuclei. Extracts of stimulated human T cells elicit chromatin binding of transcriptional activators of the interleukin‐2 (IL‐2) gene, anchoring and activity of a chromatin‐remodeling complex and hyperacetylation of the IL‐2 promoter in purified exogenous resting T‐cell nuclei. The normally repressed IL‐2 gene is transcribed in nuclei from quiescent human T cells and from various non‐T‐cell lines. This demonstrates that somatic cell extracts can be used to reprogram gene expression in differentiated nuclei. In vitro reprogramming may be useful for investigating regulation of gene expression and for producing replacement cells for the treatment of a wide variety of diseases.

Teaching cells new tricks

The direct conversion of one differentiated cell type into another--a process referred to as transdifferentiation--would be beneficial for producing isogenic (patients own) cells to replace sick or damaged cells or tissue. Adult stem cells display a broader differentiation potential than anticipated and might contribute to tissues other than those in which they reside. As such, they could be worthy therapeutic agents. Recent advances in transdifferentiation involve nuclear transplantation, manipulation of cell culture conditions, induction of ectopic gene expression and uptake of molecules from cellular extracts. These approaches open the doors to new avenues for engineering isogenic replacement cells. To avoid unpredictable tissue transformation, nuclear reprogramming requires controlled and heritable epigenetic modifications. Considerable efforts remain to unravel the molecular processes underlying nuclear reprogramming and evaluate stable of the changes in reprogrammed cells.

The regulatory landscape of osteogenic differentiation.

Differentiation of osteoblasts from mesenchymal stem cells (MSCs) is an integral part of bone development and homeostasis, and may when improperly regulated cause disease such as bone cancer or osteoporosis. Using unbiased high‐throughput methods we here characterize the landscape of global changes in gene expression, histone modifications, and DNA methylation upon differentiation of human MSCs to the osteogenic lineage. Furthermore, we provide a first genome‐wide characterization of DNA binding sites of the bone master regulatory transcription factor Runt‐related transcription factor 2 (RUNX2) in human osteoblasts, revealing target genes associated with regulation of proliferation, migration, apoptosis, and with a significant overlap with p53 regulated genes. These findings expand on emerging evidence of a role for RUNX2 in cancer, including bone metastases, and the p53 regulatory network. We further demonstrate that RUNX2 binds to distant regulatory elements, promoters, and with high frequency to gene 3′ ends. Finally, we identify TEAD2 and GTF2I as novel regulators of osteogenesis. Stem Cells 2014;32:2780–2793

Novel approaches to transdifferentiation.

Ways of directly turning a somatic cell into another (a process known as transdifferentiation) would alleviate difficulties associated with current nuclear transplantation procedures and be beneficial for producing replacement cells for therapeutic purposes. Adult stem cells have been shown to display a broader differentiation potential than anticipated and may contribute to tissues other than those in which they reside. In addition, novel transdifferentiation strategies are being developed. We illustrate here a functional reprogramming of a somatic cell using a nuclear and cytoplasmic extract derived from another somatic cell type. Reprogramming of 293T fibroblasts in an extract from T cells is evidenced by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex, changes in chromatin composition and activation of lymphoid cell-specific genes. The reprogrammed cells expressed T cell-specific surface molecules and a complex regulatory function. We propose that in vitro cell reprogramming may create possibilities for producing isogenic replacement cells for therapeutic applications. The system is also likely to constitute a powerful tool to examine the mechanisms of nuclear reprogramming as they occur in vitro.

Expression of the myodystrophic R453W mutation of lamin A in C2C12 myoblasts causes promoter-specific and global epigenetic defects

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is characterized by muscle wasting and is caused by mutations in the LMNA gene encoding A-type lamins. Overexpression of the EDMD lamin A R453W mutation in C2C12 myoblasts impairs myogenic differentiation. We show here the influence of stable expression of the R453W and of the Dunnigan-type partial lipodystrophy R482W mutation of lamin A in C2C12 cells on transcription and epigenetic regulation of the myogenin (Myog) gene and on global chromatin organization. Expression of R453W-, but not R482W-lamin A, impairs activation of Myog and maintains a repressive chromatin state on the Myog promoter upon induction of differentiation, marked by H3 lysine (K) 9 dimethylation and failure to hypertrimethylate H3K4. Cells expressing WT-LaA also fail to hypertrimethylate H3K4. No defect occurs at the level of Myog promoter DNA methylation in any of the clones. Expression of R453W-lamin A and to a lesser extent R482W-lamin A in undifferentiated C2C12 cells redistributes H3K9me3 from pericentric heterochromatin. R453W-lamin A also elicits a redistribution of H3K27me3 from inactive X (Xi) and partial decondensation of Xi, but maintains Xist expression and coating of Xi, indicating that Xi remains inactivated. Our results argue that gene-specific and genome-wide chromatin rearrangements may constitute a molecular basis for laminopathies.

Unscrambling the genomic chaos of osteosarcoma reveals extensive transcript fusion, recurrent rearrangements and frequent novel TP53 aberrations

In contrast to many other sarcoma subtypes, the chaotic karyotypes of osteosarcoma have precluded the identification of pathognomonic translocations. We here report hundreds of genomic rearrangements in osteosarcoma cell lines, showing clear characteristics of microhomology-mediated break-induced replication (MMBIR) and end-joining repair (MMEJ) mechanisms. However, at RNA level, the majority of the fused transcripts did not correspond to genomic rearrangements, suggesting the involvement of trans-splicing, which was further supported by typical trans-splicing characteristics. By combining genomic and transcriptomic analysis, certain recurrent rearrangements were identified and further validated in patient biopsies, including a PMP22-ELOVL5 gene fusion, genomic structural variations affecting RB1, MTAP/CDKN2A and MDM2, and, most frequently, rearrangements involving TP53. Most cell lines (7/11) and a large fraction of tumor samples (10/25) showed TP53 rearrangements, in addition to somatic point mutations (6 patient samples, 1 cell line) and MDM2 amplifications (2 patient samples, 2 cell lines). The resulting inactivation of p53 was demonstrated by a deficiency of the radiation-induced DNA damage response. Thus, TP53 rearrangements are the major mechanism of p53 inactivation in osteosarcoma. Together with active MMBIR and MMEJ, this inactivation probably contributes to the exceptional chromosomal instability in these tumors. Although rampant rearrangements appear to be a phenotype of osteosarcomas, we demonstrate that among the huge number of probable passenger rearrangements, specific recurrent, possibly oncogenic, events are present. For the first time the genomic chaos of osteosarcoma is characterized so thoroughly and delivered new insights in mechanisms involved in osteosarcoma development and may contribute to new diagnostic and therapeutic strategies.

Generation and Characterization of an Immortalized Human Mesenchymal Stromal Cell Line

Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use due to their capacity to self-renew and differentiate into multiple mesenchymal lineages. However, disadvantages of primary cultures of hMSCs are the limited in vitro lifespan, and the variable properties of cells from different donors and over time in culture. In this article, we describe the generation of a telomerase-immortalized nontumorigenic human bone marrow-derived stromal mesenchymal cell line, and its detailed characterization after long-term culturing (up to 155 population doublings). The resulting cell line, iMSC#3, maintained a fibroblast-like phenotype comparable to early passages of primary hMSCs, and showed no major differences from hMSCs regarding surface marker expression. Furthermore, iMSC#3 had a normal karyotype, and high-resolution array comparative genomic hybridization confirmed normal copy numbers. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding DNA methylation profiles were more diverse. The cells also had proliferation characteristics comparable to primary hMSCs and maintained the capacity to differentiate into osteoblasts and adipocytes. A detailed characterization of the mRNA and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. In summary, the immortalized mesenchymal cells represent a valuable model system that can be used for studies of candidate genes and their role in differentiation or oncogenic transformation, and basic studies of mesenchymal biology.

Reprogramming Somatic Cells for Therapeutic Applications

Directly turning a somatic cell type into another (a process referred to as transdifferentiation) would be highly beneficial for producing replacement cells for therapeutic purposes. Adult stem cells have been shown to display a broader differentiation potential than anticipated and may contribute to tissues other than those in which they reside. In addition, novel transdifferentiation strategies are being developed. We report here studies on a functional reprogramming of a somatic cell using a nuclear and cytoplasmic extract from another somatic cell type. Reprogramming of human 293T fibroblasts in an extract from a human T cell line is illustrated by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex, changes in chromatin composition, and activation of lymphoid cell-specific genes. The reprogrammed cells expressed T cell-specific surface antigens and a complex intracellular regulatory function. These studies open the door to new possibilities fo...

Reprogramming Somatic Nuclei and Cells with Cell Extracts

Publisher Summary This chapter examines procedures for reprogramming somatic nuclei and cells with cell extracts. About 293T human fibroblasts are cultured on glass coverslips. Resuspend the cells in 10 ml ice-cold cell lysis buffer (CLB). It is preferable to use a graduated 15-ml conical tube to estimate the cell volume after sedimentation. Once lysis is achieved in a tube, keep the tube on ice and proceed with all other tubes. Power and duration of sonication vary with each cell type. In order for components from the reprogramming extract to enter 293T cells, the cells must be reversibly permeabilized. Permeabilization is accomplished with the Streptococcus pyogenes toxin, streptolysin O. SLO is a cholesterol-binding toxin that forms large pores in the plasma membrane of mammalian cells. Two additional coverslips should also be used as controls for the absence of SLO. Dilute the SLO stock in ice-cold HBSS to 230 ng/ml. Expression of new proteins can also be monitored at regular intervals after the reprogramming reaction by immunofluorescence or flow cytometry using standard protocols.