Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues

Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogene myc showed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as myc might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stemcell-based approaches for liver metabolic diseases.

The role of recruited bone-marrow derived mesenchymal stem cells in the establishment of the tumor stroma and a growth-supportive environment

Jean-Paul Abbühl

The Mesenchymal Stem Cell (MSC) is a self-renewing multipotent progenitor originally found in the bone marrow. It has drawn strong interest from translational research because of the multipotency of MSCs, their immunosuppression, their intrinsic homing and their promotion of wound healing in human patients. Currently available methods rely on in vitro culture for the isolation and expansion of MSCs and most studies used these cells for their investigation. Fibroblasts constitute the connective tissue and support epithelial or hematopoietic cells. Although they have already been extensively investigated, it is not clear whether stem cells exist in vivo which specifically regenerate fibroblasts. MSCs are a potential candidate, because of their fibroblastic shape in vitro, their differentiation towards several mesodermal lineages and their supportive function. We aim to characterize the in vivo differentiation potential of MSCs toward fibroblasts in normal and tumor tissues. A new transplantation approach was developed in order to transfer labeled MSCs in the bone marrow and assess their recruitment and lineage contribution in vivo. Long-term engraftment was accomplished by isolating and transplanting these cells within one day. To this end, strategies for their purification, preconditioning of the host and transplantation methodologies were established during this thesis. We were able to enrich MSCs over 1000 fold by combining a new extraction protocol, a negative selection by MACS and a positive selection by FACS. Cloning of sorted MSCs was performed to confirm their multipotency at single cell level. GFP-expressing MSCs were able to engraft in host mice tolerant for GFP and their expansion in vivo was stimulated by block of GSK3 activity. Altogether, the chimeric MSC model was used to track the recruitment of bone-marrow derived MSCs into spontaneous breast tumors. The tumor microenvironment encompasses several non-tumorigenic cells interacting with cancer cells. Among them, cancer-associated fibroblasts are known to participate in carcinogenesis. Whilst this heterogeneous stromal population has been extensively investigated, their source(s) remain elusive. I now propose tumoral MSCs as one source of cancer-associated fibroblast, based on their ability to self-renew and reconstitute stromal heterogeneity upon serial transplantation. Finally, I introduced the new concept that the multipotency of MSCs is subjugated by cancer cells to meet their requirements for enhanced tumor progression and prepare for metastasis. I found MSCs present in the tumor microenvironment to differentiate into cancer-associated osteoblasts and produce hydroxyapatite-mineralization in vivo. Microcalcification is an accepted poor prognostic feature in breast cancer patients but its function and origin is not documented. I showed that hydroxyapatite was able to promote proliferation of tumor cells and increased intracellular calcium concentration. Lastly, cancer-associated osteoblast and hydroxyapatite were able to promote tumor growth and metastasis in vivo. To conclude, bone marrow derived MSCs are circulating stem cells that participate in the establishment of the pathogenic stroma in breast cancer. Owing to their multipotency, MSCs give rise to cancer-associated osteoblasts that induce pro-malignant mineralization in situ. Subsequently, cancer cells harbor increased intracellular calcium concentration, enhanced proliferation and metastasis. We foresee the development of new stroma-targeted therapies that prevent the mineralization of tumor tissue and reduce metastasis in breast cancer patients.

EPFL2014

Studies on lentiviral-mediated transgenesis

Marc-Olivier Sauvain

Transgenic animals are essential research tools, whether to address basic biological questions or to develop preclinical models of human diseases. Their generation through the injection of naked plasmid DNA into the male pronucleus of a fertilized oocyte has been the standard practice for almost 3 decades. However, this approach is invasive, largely limited to the mouse and results in low rates of transgene integration. Although the inefficiency of pronuclear injection can be overcome in small animals by high-throughput screening for transgene integration, this becomes economically more challenging in larger animals, such as sheep, pigs or cows, because of the extreme costs associated with this procedure (

60'000-

300'000). Thus, new strategies to enhance the production and the variety of transgenic animals would be an important asset. One such approach has emerged through the use of lentiviral vectors, a gene delivery system that can efficiently transfer and stably integrate its cargo into a wide variety of cell types from numerous species, and has been successfully applied to generate transgenic mice, rats, pigs and cows. The increasing use of lentivector-mediated transgenesis warrants a full analysis of its modalities. As a step towards this goal, the present study aimed at characterizing the genotypic features of transgenic mice generated by lentiviral infection of zygotes. First, as an indirect method to measure the kinetics of lentiviral integration in early embryos, we examined the rates of transmission of individual proviruses from G0 transgenic mice to their G1 progeny using a PCR-based technique specific for each integrant. The mean rate of transmission of 44% for individual proviruses suggests that vector integration was most often established between the post-S phase of the one-cell stage and pre-S phase of the two-cell stage. We then moved on to define proviral integration site selection in lentivector-generated transgenic mice. Using LAM-PCR-mediated amplification and sequencing of the host genome-provirus junctions, we found that the frequency of proviral integration inside a gene was 1.75- and 1.88-fold higher in transgenic mice and 3T3 fibroblasts, respectively, than expected from the distribution of annotated genes in the mouse genome (Pmice .03e 09; P3T3 5.55e 16). Moreover, integration was not influenced by the transcriptional orientation of the target gene and tended to favour the middle of transcribed regions. We also observed a subtle difference in the integration patterns of lentiviral vectors in 3T3 fibroblasts and transgenic mice, with the latter exhibiting a slightly higher frequency of integration into repeats. Although this difference was statistically not significant, it might reflect the elevated transcriptional activity of repetitive elements in preimplantation embryos. Since proviruses favour intragenic localization, we analyzed G2 mice homozygous for specific lentiviral integrants using PCR-based techniques and observed that the frequency of homozygosity was below the expected mendelian transmission rate of 25%. This suggested that some of the homozygous G2 mice must be nonviable due to the potential of retrovirus integrants to inactivate target genes. However, the normal rates of G1 representation of proviruses that did not achieve homozygosity in G2 suggested an absence of toxicity in the heterozygous state. Processes governing the earliest steps of mammalian development are still incompletely understood. In particular, the stage at and modalities by which early blastomeres become committed to either the inner cell mass (ICM), which subsequently yields the embryo proper, or the trophectoderm, which serves as precursor of extra-embryonic tissues, is unclear. The long held "random" hypothesis for development of the embryonic-abembryonic axis states that, within these two structures, early blastomeres are equally represented. However, recent lineage-tracing experiments support a model of "developmental bias", in which the progeny of the early blastomeres is allocated in a biased fashion. In our study, we took advantage of the specific integration pattern of lentiviral vector – i.e. each lentiviral vector integrates the host genome at specific sites – to "fingerprint" individual blastomeres and to follow them during embryogenesis. For this, we injected lentiviral vectors into embryos at either the one-cell or the two-cell stage. The Southern Blot (SB) analysis performed at E12.5 revealed that the infection at the one-cell stage yielded integrants shared for their majority by both embryonic and extra-embryonic tissues (74% of similar bands), while the infection at the two-cell stage induced a drastically different picture, with only a minority of common integrants between both types of tissues (22% of similar bands). This suggests that early blastomeres – from the two-cell stage on – contribute differentially to these lineages, which argues against a purely stochastic model. However, we still obtained 22% of shared integrations after infection at the two-cell stage, with only 8 out of 27 embryos showing a complete segregation of the proviral integration patterns. This rules out a "fully determined" model. Thus, our observations rather support the "developmental bias" hypothesis.

EPFL2009

Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency

Patrick Schorderet

The therapeutic potential of embryonic stem cells (ESc) is a key scientific interest, but the ethical implications of stem cell research have been controversial and are often a major detriment to the advancement of our knowledge. Recently, several landmark studies both in mouse and human have defined a set of four transcription factors (Klf4, c-Myc, Oct4 and Sox2) whose ectopic expression enables a somatic nucleus to shift back to a pluripotent state. These cells, referred to as induced pluripotent stem (iPS) cells, not only express markers characteristic of pluripotent cells such as alkaline phosphatase, SSEA1, Nanog and Oct4, but both their epigenetic state and their biological potency have been shown to be indistinguishable from those of ES cells. Although these results have been a great breakthrough in stem cell research and have brought hope for an alternative to embryo-derived ES cells, skeptics argue that these pluripotent cells derive from a small subset of remaining multipotent cells or cells that are more amenable to direct reprogramming. Following this approach, a fundamental question remains unsolved: namely, whether this process holds true in terminally differentiated cells. Also, clarifying whether cells of a distinct lineage are equally amenable to direct reprogramming throughout their developmental stages would determine if distinct prerequisites are needed and would be an important step towards understanding the biological processes involved in this transformation. Here, we used transgenic and inducible expression vectors of the previously described factors to reprogram mouse B lymphocytes. Alone, the four factors were able to convert non-terminally differentiated pro-B cells into a pluripotent state but failed to do so in cells that had undergone full B cell receptor rearrangements. Instead, expression of a myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα), known to disrupt the epigenetic faith of B cells, was shown to be required. Multiple iPS lines were derived and characterized using various methods. Ultimately, they were shown to be fully pluripotent by their contribution to germline transmission. Our study provides definitive proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency

2008